A lipid plug affects K2P6.1(TWIK-2) function

A lipid plug affects K2P6.1(TWIK-2) function | bioRxiv /* */ /* */ <!-- <!-- /*! * yepnope1.5.4 * (c) WTFPL, GPLv2 */ (function(a,b,c){function d(a){return"[object Function]"==o.call(a)}function e(a){return"string"==typeof a}function f(){}function g(a){return!a||"loaded"==a||"complete"==a||"uninitialized"==a}function h(){var a=p.shift();q=1,a?a.t?m(function(){("c"==a.t?B.injectCss:B.injectJs)(a.s,0,a.a,a.x,a.e,1)},0):(a(),h()):q=0}function i(a,c,d,e,f,i,j){function k(b){if(!o&&g(l.readyState)&&(u.r=o=1,!q&&h(),l.onload=l.onreadystatechange=null,b)){"img"!=a&&m(function(){t.removeChild(l)},50);for(var d in y[c])y[c].hasOwnProperty(d)&&y[c][d].onload()}}var j=j||B.errorTimeout,l=b.createElement(a),o=0,r=0,u={t:d,s:c,e:f,a:i,x:j};1===y[c]&&(r=1,y[c]=[]),"object"==a?l.data=c:(l.src=c,l.type=a),l.width=l.height="0",l.onerror=l.onload=l.onreadystatechange=function(){k.call(this,r)},p.splice(e,0,u),"img"!=a&&(r||2===y[c]?(t.insertBefore(l,s?null:n),m(k,j)):y[c].push(l))}function j(a,b,c,d,f){return q=0,b=b||"j",e(a)?i("c"==b?v:u,a,b,this.i++,c,d,f):(p.splice(this.i++,0,a),1==p.length&&h()),this}function k(){var a=B;return a.loader={load:j,i:0},a}var l=b.documentElement,m=a.setTimeout,n=b.getElementsByTagName("script")[0],o={}.toString,p=[],q=0,r="MozAppearance"in l.style,s=r&&!!b.createRange().compareNode,t=s?l:n.parentNode,l=a.opera&&"[object Opera]"==o.call(a.opera),l=!!b.attachEvent&&!l,u=r?"object":l?"script":"img",v=l?"script":u,w=Array.isArray||function(a){return"[object Array]"==o.call(a)},x=[],y={},z={timeout:function(a,b){return b.length&&(a.timeout=b[0]),a}},A,B;B=function(a){function b(a){var a=a.split("!"),b=x.length,c=a.pop(),d=a.length,c={url:c,origUrl:c,prefixes:a},e,f,g;for(f=0;f<d;f++)g=a[f].split("="),(e=z[g.shift()])&&(c=e(c,g));for(f=0;f<b;f++)c=x[f](c);return c}function g(a,e,f,g,h){var i=b(a),j=i.autoCallback;i.url.split(".").pop().split("?").shift(),i.bypass||(e&&(e=d(e)?e:e[a]||e[g]||e[a.split("/").pop().split("?")[0]]),i.instead?i.instead(a,e,f,g,h):(y[i.url]?i.noexec=!0:y[i.url]=1,f.load(i.url,i.forceCSS||!i.forceJS&&"css"==i.url.split(".").pop().split("?").shift()?"c":c,i.noexec,i.attrs,i.timeout),(d(e)||d(j))&&f.load(function(){k(),e&&e(i.origUrl,h,g),j&&j(i.origUrl,h,g),y[i.url]=2})))}function h(a,b){function c(a,c){if(a){if(e(a))c||(j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}),g(a,j,b,0,h);else if(Object(a)===a)for(n in m=function(){var b=0,c;for(c in a)a.hasOwnProperty(c)&&b++;return b}(),a)a.hasOwnProperty(n)&&(!c&&!--m&&(d(j)?j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}:j[n]=function(a){return function(){var b=[].slice.call(arguments);a&&a.apply(this,b),l()}}(k[n])),g(a[n],j,b,n,h))}else!c&&l()}var h=!!a.test,i=a.load||a.both,j=a.callback||f,k=j,l=a.complete||f,m,n;c(h?a.yep:a.nope,!!i),i&&c(i)}var i,j,l=this.yepnope.loader;if(e(a))g(a,0,l,0);else if(w(a))for(i=0;i (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0];var j=d.createElement(s);var dl=l!='dataLayer'?'&l='+l:'';j.src='//www.googletagmanager.com/gtm.js?id='+i+dl;j.type='text/javascript';j.async=true;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-M677548'); Skip to main content Home About Submit ALERTS / RSS Search for this keyword Advanced Search New Results A lipid plug affects K 2P 6.1(TWIK-2) function Abhisek Mondal , Sangeeta Niranjan , View ORCID Profile Daniel L. Minor Jr doi: https://doi.org/10.1101/2025.06.11.659167 Abhisek Mondal 1 Cardiovascular Research Institute Find this author on Google Scholar Find this author on PubMed Search for this author on this site Sangeeta Niranjan 1 Cardiovascular Research Institute Find this author on Google Scholar Find this author on PubMed Search for this author on this site Daniel L. Minor Jr 1 Cardiovascular Research Institute 3 Departments of Biochemistry and Biophysics, and Cellular and Molecular Pharmacology 4 California Institute for Quantitative Biomedical Research 5 Kavli Institute for Fundamental Neuroscience University of California , San Francisco, California 93858-2330 USA 6 Molecular Biophysics and Integrated Bio-imaging Division Lawrence Berkeley National Laboratory , Berkeley, CA 94720 USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Daniel L. Minor Jr For correspondence: daniel.minor{at}ucsf.edu Abstract Full Text Info/History Metrics Supplementary material Preview PDF Abstract Lipids are integral to ion channel function yet delineating mechanisms by which they affect function remains challenging. Within the K 2P family of leak potassium channels 1 – 3 , observation of tubular densities interpreted as alkyl chains occupying lateral fenestrations linking the pore and bilayer 4 – 8 raised the possibility that lipid access from the bilayer acts as a regulatory mechanism 4 – 7 . Here, we present cryo-electron microscopy (cryo-EM) structures of the human leak potassium channel K 2P 6.1 (TWIK2) 9 – 11 and mutants in nanodisc and detergent environments that reveal an unusual conformation in the first selectivity filter (SF1) and a pair of two-chain lipids within the channel cavity (denoted the ‘lipid plug’). The chains of each plug lipid occupy separate binding sites that laterally extend to the bilayer from the channel cavity. One, the upper leg, matches the previously identified alkyl chain binding site 4 – 8 , 12 . The second, the lower leg, occupies a fenestration common with K 2P 1.1 (TWIK1) 13 . Together, they demonstrate a bidentate means to coordinate each plug lipid that offers a reinterpretation of previous observations. Structures of a K 2P 6.1 (TWIK2) mutant that directs the channel to the plasma membrane 14 and an R257A mutant that increases function yield plugged and unplugged forms. Notably, the R257A plugged form shows a change in lipid plug position, indicating a key role for this residue in lipid binding. Together, our data suggest that occupation of the central cavity by the lipid plug serves as a mechanism to render the TWIK channels inactive and points to the importance of lipid plug removal to create an ion permeable pore. Such a mechanism could provide a potent way for limiting the leak function of K 2P s based on cellular location or other contextual factors. Introduction K 2P potassium channels produce leak currents that are important for controlling resting membrane potential in diverse cell types 1 – 3 . Fifteen human K 2P channel subunits comprise six subfamilies (TWIK, TREK, THIK, TASK, TALK, and TRESK) that each respond to diverse sets of physiological cues including physical force, lipid modulation, and signals from various signaling cascades 15 . Structural studies of members from the TWIK 4 , 5 , TREK 8 , 16 , 17 , TALK 18 , TASK 19 , and THIK 20 , 21 subfamilies reveal a shared architecture 15 in which the two pore domains (PD1 and PD2) of an individual K 2P s subunit form a pseudo tetrameric pore upon subunit dimerization. In contrast to other types of potassium channels, the selectivity filter (SF) ‘C-type’ gate acts as the principal site of K 2P modulation for most K 2P s 15 , 16 , 22 – 26 . Despite their relatively small size (∼60-70 kDa), K 2P s contain a wealth of binding sites for various classes of lipid and small molecule modulators whose roles in channel gating remain imperfectly understood 15 . In particular, the potential role of lipid access through a lateral fenestration below the selectivity filter has attracted much attention 5 , 6 , but whether this represents a mechanism for direct block of the pore remains a point of controversy as simulations indicate that even though lipid tails can enter they do not block the pore 13 , 27 . K 2P 6.1 (TWIK2) 9 – 11 belongs to the TWIK subfamily comprising K 2P 1.1 (TWIK1), K 2P 6.1 (TWIK2), and K 2P 7.1 2 and is found in the gastrointestinal tract, vasculature, and immune system 9 where it is linked to vascular 28 and pulmonary 29 hypertension and sepsis responses 30 . K 2P 6.1 (TWIK2) and K 2P 1.1 (TWIK1) are notable for their endolysomal localization 31 . Functional and cell biology studies indicate that in K 2P 6.1 (TWIK2) this intracellular localization originates from internalization signals that favor this location and largely prevent plasma membrane expression 14 . Interestingly, in macrophages transit of endosomally sequestered K 2P 6.1 (TWIK2) to the plasma membrane following a signal initiated by extracellular ATP has been implicated in the process underlying NLRP3 ( N OD, L RR and p yrin domain-containing protein 3 ) inflammasome activation 30 , 32 – 34 . This K 2P 6.1 (TWIK2) function parallels that described for the THIK subfamily in NLRP3 activation in microglia and interleukin 1β (IL–1β) release 35 – 37 , highlighting the importance of the link between K 2P cellular location and function. Although K 2P 1.1 (TWIK-1) has been structurally characterized 4 , 5 , 18 , the structure of K 2P 6.1 (TWIK2) has not been previously described. Here, we report structural studies of human K 2P 6.1 (TWIK2) and mutants in lipid nanodisc and detergent environments. The data reveal a channel architecture that largely resembles K 2P 1.1 (TWIK1) having three notable features: an extra helix in the extracellular cap domain (the ‘ear helix’), an unusual conformation of a key residue in the first selectivity filter (SF1), and block by pair of intracellular lipids that engage the channel through a set of bidentate interactions with lateral fenestrations linking the pore and bilayer. Elements from this last feature occupy the site that has been identified in K 2P 1.1 (TWIK1) 4 , 5 and other K 2P s 6 – 8 as binding site for a single alkyl chain 4 , 6 – 8 or detergent 4 , 12 . Structures of a previously characterized K 2P 6.1 (TWIK2), a mutant that increases the plasma membrane localization and activity 14 , yield plugged and unplugged forms that highlight the importance of lipid removal for channel function. Moreover, structure of the R257A mutant that enhances function and affects a key lipid coordinating residue reveals a descent of the lipid plug towards the intracellular opening, highlighting the important role of Arg257 in lipid coordination. The fact that the lipid plug forms a physical barrier that prevents ion passage and that this plug can be destabilized by mutations that enhance channel function suggests that the function of the TWIK subfamily depends on lipid plug removal. Results Human K 2P 6.1(TWIK-2) structure reveals an unusual selectivity filter conformation and a lipid plug Expression and purification of full-length human K 2P 6.1(TWIK-2) (denoted ‘TWIK2’) from HEK293 cells yielded a sample suitable for cryo-EM studies (Fig. S1a) that we used for structural characterization in MSP1E1 38 lipid nanodiscs under high potassium (200 mM KCl) conditions (Figs. S1 and S2, Table 1 ). The structure (TWIK2:ND) has an overall resolution of 3.2Å, with the best-defined parts reaching 2.2Å ( Fig. 1a and S2c, Table 1 ). TWIK2 has the canonical architecture found throughout the K 2P family 15 (Fig. S3) and is very similar to its related TWIK subfamily member, K 2P 1.1 (TWIK-1) 4 , 5 (RMSD Cα = 0.817Å 3UKM 4 , 1.836Å 7SK0 5 , 1.338Å 7SK1 5 ). The two PDs from each subunit are arranged around the central axis of the channel with the M2 and M4 helices lining the pore and the M1 and M3 helices located on the periphery. The M1 helix is domain swapped between the two subunits and connects to the extracellular Cap domain as in other K 2P s 15 . There is a branched aqueous pathway, the extracellular ion pathway (EIP), between the Cap and extracellular face of the pore that connects the external mouth of the channel selectivity filter (SF) with the extracellular space 4 , 7 , 15 . Different from other K 2P s, the TWIK2 Cap domain has a third helix (denoted the ‘ear helix’) that connects the Cap to the loop leading to the P1 helix ( Fig. 1b , S4a-b). Ear helix residues Gly73, Val76, Leu77, and Asn79 from one subunit pack against the α2 helix from its own Cap and the α1 and α2 Cap helices from its dimeric partner. These positions are largely conserved among vertebrate TWIK2s (Fig. S5) and with exception of a hydrogen bond pair (Glu41-Asn79) are conserved in TWIK1 (Fig. S4b). By contrast, these sites are poorly conserved in other K 2P classes (Figs. S4a-b), indicating that the Ear helix is a feature of the TWIK subfamily. Evaluation of surface electrostatics highlights the electronegative character of the extracellular EIP, a prominent positively charged surface on the intracellular M2-M3 loop, and a relatively hydrophobic inner cavity bounded by the selectivity filter and Arg257 (Fig. S6). Download figure Open in new tab Figure 1 K 2P 6.1(TWIK-2) structural features. a, K 2P 6.1(TWIK-2) cartoon diagram (deep teal and bright orange) showing side (left and center) and intracellular (right) views. Lipid plug (grey) is shown in space filling. Grey bars indicate membrane. Potassium ions are shown as purple spheres. b, Comparison of K 2P 6.1(TWIK-2) SF1 (left) and SF2 (right) filter conformations. Select matching positions are shown as sticks. c , Sequence comparison P1, SF1, and initial segment of M2 from PD1 for K 2P 6.1 (TWIK-2) with other K 2P s. Red oval indicates Tyr111 site. Labels indicate residues that interact with Tyr111 in the ‘down’ state. Conservation is indicated in blue. Tyr111 and similar residues are shaded red. Sequences are for human: K 2P 6.1(TWIK-2) (GENBANK 4758624); K 2P 1.1(TWIK-1) (GENBANK 4504847); K 2P 13.1(THIK-1) (GENBANK 16306555); K 2P 12.1(THIK-2) (GENBANK 11545761); K 2P 2.1(TREK-1) (GENBANK 14589851); K 2P 10.1(TREK-2) (GENBANK 20143944); K 2P 4.1(TRAAK) (GENBANK 15718767); K 2P 3.1(TASK-1) (GENBANK 4504849); K 2P 9.1 (TASK-3) (GENBANK 542133161); K 2P 15.1(TASK-5) (GENBANK 333440483); K 2P 5.1(TASK-2) (GENBANK 333440483); K 2P 16.1(TALK-1) (GENBANK 14149764); K 2P 17.1(TALK-2) (GENBANK 17025230); and K 2P 18.1(TRESK) (GENBANK 32469495). d , K 2P 13.1(THIK-1) Try111 environment and interactions. ‘up’ (bright orange) and ‘down’ (olive) Tyr111 conformations are indicated. e, Cryo-EM density for the region including Tyr111 (σ=2.5). View this table: View inline View popup Download powerpoint Table 1 Cryo-EM data collection, refinement and validation statistics Consistent with structure solution in high potassium conditions, the TWIK2 selectivity filter shows density for ions at the S1-S4 positions as well as at the extracellular S0 position ( Figs. 1b and e , S2b). The selectivity filters of K 2P differ from some canonical features seen in other potassium channels. Notably, diverse amino acids occupy the last position of SF1 potassium channel signature selectivity filter sequence ‘TxTTxGYG D ’ 15 , 39 ( Fig. 1c ), and contrast SF2 where this site is strongly conserved as an aspartic acid (Fig. S7a) as found in homomeric potassium channels 40 . Despite the SF1 sequence variation, in most K 2P structures 15 , both SFs adopt essentially the same local conformations in which the sidechain of the residue following the last SF glycine interacts with a network of residues behind the selectivity filter (Fig. S7b), similar to homomeric potassium channels 41 . SF1 in TWIK2 stands out by having a large aromatic residue, Tyr111, following the selectivity filter ‘GYG’ sequence ( Fig. 1c ). The structure shows that rather than adopting the canonical ‘down’ conformation seen in SF1 from K 2P 1.1 (TWIK1) 4 , 5 and K 2P 2.1(TREK-1) 16 exemplars (Fig. S7b-c) and the equivalent SF2 position ( Fig. 1c , Fig. S7b and d), Tyr111 occupies an ‘up’ conformation in which it interacts with Arg71 and Arg74 from the ear helix base ( Fig. 1b-d ). Inspection of the TWIK2:ND density also showed evidence for a second Tyr111 conformation corresponding to the ‘down’ state ( Fig. 1d-e ) in which Tyr111 interacts with a pocket formed by P1 helix residues Phe98, Phe99, and Thr102 and SF1 residue Tyr109 ( Fig. 1d ). The ‘up’ and ‘down’ conformations are essentially Tyr111 rotamer changes that do not require major changes in the surrounding SF structure. Notably, a similar ‘up’ rotamer of the corresponding residue in K 2P 3.1 (TASK-1), His98, was observed in a pH 7.5 structure (Fig. S7e) 42 contrasting a ‘down’ conformation seen at higher pH 43 . Together, these observations show that the SF1 ‘up’/’down’ rotamer change can occur in different K 2P subfamilies. The additional conformational changes observed for the equivalent SF1 position in K 2P 1.1 (TWIK1) 5 (Fig. S7f) and K 2P 2.1(TREK-1) 25 under conditions associated with inactivation of the channel C-type SF gate highlight the importance of structural changes this site for K 2P function. The TWIK2 structure also revealed a second notable feature, a pair of branched densities that correspond to the legs two lipids stacked face to face that obstruct the intracellular aqueous cavity ( Figs. 1a , 2a , S2a-d). The two legs of each lipid fill the hydrophobic inner cavity above Arg257 and occupy two fenestrations at the interface between P2 and M4 from one subunit and M2 from the other that lead to the membrane bilayer. The upper leg site formed by P2, M2 and M4 is lined by Leu128, Pro132, Met135, Leu246, Met249, Val250, and Leu253. This site corresponds to a site occupied by a single alkyl chain in TWIK1 4 ( Figs. 2b-d , S8a). The lipid lower leg site is formed by M2 and M4 and is lined by Leu136, Leu253, Arg257, Ser260, Thr266, and Leu270 ( Figs. 2b-d , S8a). The Arg257 sidechain, a residue conserved in vertebrate TWIK2 (Fig. S5), directly contacts the lipid glycerol backbone ( Figs. 2c , S8a). Except for Arg257, the lipid plug contacting residues are conserved between TWIK1 and TWIK2 (Fig. S8b-c). Download figure Open in new tab Figure 2 K 2P 6.1(TWIK-2) lipid plug blocks the central cavity. a, K 2P 6.1(TWIK-2) cartoon diagram (deep teal and bright orange). Cryo-EM density for lipid plug (grey) is shown (σ =3.0). Inset shows slice through space-filling model of the channel, lipid plug cryo-EM density and stick representation of lipid plug. b, Superposition of K 2P 6.1(TWIK-2) (deep teal and bright orange) and K 2P 1.1(TWIK-1) (magenta) (PDB:3UKM) 4 . K 2P 6.1(TWIK-2) lipid plug (grey and black) is shown as sticks and semi-transparent space filling. Upper and lower legs for one lipid are indicated. TWIK1 Alkyl chain 4 is shown as sticks. c, K 2P 6.1(TWIK-2) lipid plug interactions. One lipid plug chain is shown (black, space filling). Contacting residues from the two K 2P 6.1(TWIK-2) subunits (deep teal and bright orange) are shown as sticks. d, View from the center of the bilayer towards the upper and lower leg binding sites. Lipid plug chains are grey and black and shown in space filling. Interacting residues are shown as sticks. e, Lipid plug density (1.9σ) from K 2P 1.1 (TWIK-1) at pH5.5 (EMDB 25169) 5 . Model shows a single K 2P 6.1(TWIK-2) subunit and lipid plug (grey). The presence of the lipids in the channel cavity prompted us to ask whether these cavity-filling molecules originated from the nanodisc reconstitution step or were present from an earlier step in channel purification. Hence, we determined the structure of TWIK2 in detergent at 3.7Å (local resolution to 2.2Å) (TWIK2:Det) (Figs. S9-S10, Table 1 ). TWIK2:Det was essentially identical to TWIK2:ND (RMSD Cα =0.44Å) having the same features, including an ‘up’ Tyr111 (Fig. S10a) and ions at sites S0-S4 of the selectivity filter. Importantly, the structure shows density corresponding to the two lipid molecules forming the lipid plug (Fig. S10a), demonstrating that these entities do not originate from the lipids used for the nanodisc reconstitution but come from the cells used to produce the protein sample. The TWIK1 structure 4 also has a fenestration below the upper leg site 13 . Comparison with the lipid plug from our TWIK2 structure shows that this cavity matches the position of the plug lower leg (Fig. S8d), suggesting that TWIK1 can accommodate lipids using theTWIK2 bi-legged lipid plug binding pose. Prompted by this structural similarly, we inspected previously reported cryo-EM densities for TWIK1 5 for evidence of the lipid plug. Remarkably, the density map of TWIK1 at pH 5.5, a structure thought to represent an inactivated channel, shows a bifurcated density in the channel central cavity that is well matched by the TWIK2 lipid plug ( Fig. 2e ). The map for the TWIK1 pH 7.4 structure, in which the upper site was proposed to be empty of the alkyl chain shows a weaker version of a similar density (Fig. S8e). Thus, the lipid plug binding mode appears to be shared among members of the TWIK subfamily. These observations suggest a revised interpretation of the origin of the alkyl chain density in the upper site of TWIK1 and indicate that it is from one of two legs of a lipid that blocks the pore as we observe in TWIK2. The conservation of the residues lining both lipid-occupied cavities, particularly in M4, does not extend to other K 2P family members (Fig. S8c). Hence, the architecture that enables this bifurcated type of lipid binding in the channel central cavity appears to be a special feature of the TWIK subfamily. Together, these results highlight the unique binding mode of theTWIK2 plug lipid in which each lipid is anchored in the central cavity by occupying the upper and lower leg sites on opposite sides of the channel. A trafficking mutation yields unplugged channels TWIK2 is notable for poor functional expression unless it is directed to the plasma membrane by mutations in lysosomal targeting motifs 14 . To test whether this localization change might affect channel structure or the lipid plug, we determined the structure of a previously characterized TWIK2 mutant 14 , 33 , 34 , denoted TWIK2 RM (for ‘Retention Mutant’), that is predominantly found at the plasma membrane as a result of a set of C-terminal tail mutations (I289A/L290A/Y308A) 14 . Initial cryo-EM analysis of TWIK2 RM showed one form corresponding to the lipid bound state (Fig. S11). However, based on the functional effects of the TWIK2 RM mutations 14 , we decided to pursue further classification using a mask focused on the central cavity to determine if we could identify a form representing a functional channel. This analysis yielded two channel forms at 3.9Å resolution each (Fig. S11, Table 1 ) that we denoted as TWIK2 RM -Plugged and TWIK2 RM -Unplugged due to the presence or absence of the lipid plug. The structure of TWIK2 RM -Plugged was essentially identical to TWIK2:Det (RMSD Cα =0.7Å) ( Fig. 3a ), showed Tyr111 in the up position (Fig. S12a), and two plug lipids blocking the central channel cavity ( Fig. 3b-c , S12a). TWIK2 RM -Uplugged was also overall very similar to TWIK2:Det (RMSD Cα =0.7Å) ( Fig. 3d ) and had Tyr111 from SF1 in the up position (Fig. S13a). Notably, the plug lipid density was absent ( Fig. 3f , S13b). Instead, we found a spherical density just below the S4 position of the selectivity filter that we assigned as the ‘cavity ion’ ( Figs. 3d-e , S13a), reminiscent of results seen for a structure of K 2P 4.1 in the absence of its fenestration density 6 . Following the identification of the two forms, we reprocessed the TWIK2:ND and TWIK2:Det data using the same strategy used to obtain plugged and unplugged form of TWIK2 RM but failed find a class corresponding to the unplugged form. These data suggest that the strong intracellular retention of the wild-type channel is associated with the lipid plugged form, whereas some fraction of the channel lacks this plug when the channel can reach the plasma membrane. The region bearing the trafficking mutations is not seen in any of our structures, suggesting that this region is disordered. The density for the ions in the selectivity filter of both the TWIK2 RM plugged (Fig. S12a) and TWIK2 RM unplugged (Fig. S13a) structures suggests differences in ion occupancy at the S0 and S1 sites from the TWIK2 structures. The structural analysis of the trafficking mutant TWIK2 RM shows that the channel can exist in two states, one having two lipid plug molecules (TWIK2:ND, TWIK2:Det, and TWIK2 RM :Det plugged) and one in which access to the central cavity is unhindered (TWIK2 RM :Det unplugged). Comparison of representative structures for the plugged (TWIK2:ND) and unplugged (TWIK2 RM :Det unplugged) forms shows that the two plug lipids effectively block ion access to the channel by filling the central intracellular cavity below the selectivity filter ( Fig. 3g-h ). Download figure Open in new tab Figure 3 Comparison of plugged and unplugged K 2P 6.1 (TWIK2) structures. a , Superposition of TWIK2:ND (deep teal and bright orange) and TWIK2 RM :Det (plugged) (deep purple) structures. b, Close up view of the central cavity from ‘a’. Lipid plugs from TWIK2:ND and TWIK2 RM :Det (plugged) are shown as spheres (grey) and sticks (deep purple), respectively. c, Intracellular view of a slice through the TWIK2 RM :Det (plugged) density. Subunits are deep teal and bright orange, lipid plug density is grey. d, Superposition of TWIK2:ND (deep teal and bright orange) and TWIK2 RM :Det (unplugged) (firebrick) structures. d, Close up view of the central cavity from ‘d’. Lipid plug from TWIK2:ND is shown as spheres (grey). TWIK2 RM :Det (unplugged) cavity ion is labeled. f, Intracellular view of a slice through the TWIK2 RM :Det (unplugged) density. Subunits are firebrick and light red. g, Pore profile of K 2P 6.1(TWIK2) (deep teal and bright orange) calculated using HOLE 53 . Selectivity filter and key cavity positions are shown as sticks. h, K 2P 6.1(TWIK2) pore profiles for plugged (blue) and unplugged (magenta) forms. Selectivity filter (SF) (grey) and cavity (blue) are indicated. in ‘g’ and ‘h’. Functional studies highlight the importance of lipid-interacting cavity residues and Tyr111 We set out to test key features identified by the TWIK2 structures. As previously reported 10 , 14 , TWIK2 did not produce measurable currents in Xenopus oocytes using two electrode voltage clamp (TEVC), whereas mutation of the endolysosomal targeting signal (I289A/L290A/Y308A) 14 (TWIK2 RM ) yielded readily measurable TWIK2 channels (Fig. S14a) that provided a platform for testing the effects of mutants. To test the importance of the lipid plug contacts, we examined the effects of mutations at lipid plug-channel at three places along the channel central axis ( Fig. 4a-c ), with a focus on mutation to negatively charged residues to destabilize lipid-channel contacts. V131D, located near the base of the selectivity filter, did not make functional channels ( Figs. 4b-c , S14a). However, introduction of acidic residues at Met135 (M135D) and Arg257 (R257E) resulted increased basal activity with respect to TWIK2 RM , as did replacement of Arg257 by alanine (R257A) ( Fig. 4b-c , S14a). Comparison of ion selectivity by following the reversal potential as a function of external potassium showed no significant differences in the functional mutants from TWIK2 RM (Fig. S14b). The increased activity observed for mutation at sites that interact with the blocking lipids is consistent with the idea that removal of the lipid plug is key to channel function. Further the fact that introduction of a negative charge and truncation to alanine at the Arg257 site increases activity points to an important role for this position in TWIK2 function. Download figure Open in new tab Figure 4 K 2P 6.1(TWIK2) cavity and filter sites affect function. a, View of the K 2P 6.1(TWIK2) lipid plug from center of the membrane. Lipid plug is shown as grey spheres. Key interacting residues are shown as sticks. Potassium ions are purple spheres. b, Exemplar current-voltage responses from TEVC Xenopus oocytes expressing the indicated channels and cavity mutants. Uninjected (grey), TWIK2 (sky blue), TWIK2 RM (blue), TWIK2 RM V131D (olive), TWIK2 RM M135A (orange), TWIK2 RM M135A (red), TWIK2 RM R257A (yellow), and TWIK2 RM R257E (lavender). Inset shows protocol. c, Effects of mutants on K 2P 6.1(TWIK2) basal activity at 0 mV. **** p <0.0001, *** p <0.001, ** p < 0.01, * p = 0.01 -0.05. Statistical analysis was performed using a One-Way ANOVA and multiple comparisons were performed with Tukey’s test. Error bars are S.E.M.. d, Exemplar current-voltage responses from TEVC Xenopus oocytes expressing the indicated channels and mutants. Uninjected (grey), TWIK2 (sky blue), TWIK2 RM (blue), TWIK2 RM Y111A (green), TWIK2 RM Y111H (salmon), and TWIK2 RM Y111N (maroon). Inset shows protocol. e, Effects of mutants on K 2P 6.1(TWIK2) basal activity at 0 mV. **** p <0.0001, *** p <0.001, ** p < 0.01, * p = 0.01 -0.05. Statistical analysis was performed using One-Way ANOVA and multiple comparisons were performed with Tukey’s test. Error bars are S.E.M.. The fact that Tyr111 is the largest residue seen at this filter position ( Fig. 1c ) and was observed in both up and down conformations prompted us to test effect of replacing this residue with smaller, more commonly observed amino acids or with alanine. Replacement of this position with the most common residue found at this site in other K 2P s, asparagine (Y111N) ( Fig. 1c ) caused a reduction in basal current, as did replacement with alanine (Y111A). By contrast, Y111H yielded basal currents similar to wild type. Despite the fact that the mutation site is in the selectivity filter, none of these changes affected responses to external potassium (Fig. S14d) indicating that the selectivity of the channels remained intact. Notably, all three changes resulted in channels that showed inactivation (Fig. S14 a and c) supporting the importance of Tyr111 in the function of the K 2P 6.1 (TWIK2) selectivity filter gate. R257A mutation changes lipid plug position To investigate the structural consequences of perturbing lipid-coordinating residues ( Fig. 4c ), we determined the structure of TWIK2 RM R257A, a mutant that increases TWIK2 RM basal currents (Fig. S15). As with TWIK2 RM , cryo-EM analysis of single particles of TWIK2 RM R257A using focused classification on the central cavity yielded two channel classes, one having the lipid plug, TWIK2 RM R257A-plugged (3.9Å resolution), and one in which the plug was absent, TWIK2 RM R257A-unplugged (4.1Å resolution) (Figs. S15-S17, Table 1 ). The overall structures of both TWIK2 RM R257A forms were similar to the TWIK2:ND structure ( Fig. 5a-d ) (RMSD Cα =0.82Å). Both had Tyr111 in the up position (Figs. S16a and S17a). TWIK2 RM R257A-plugged showed clear ion density at selectivity filter sites S0-S4 (Fig. S16a), whereas there were fewer well-defined ions in the TWIK2 RM R257A-unplugged form (Fig. S17a). Strikingly, in the TWIK2 RM R257A-plugged form, the lipid plug resides ∼5Å closer to the intracellular opening of the channel than in the other plugged TWIK2 structures ( Figs. 5a-b , and S18a). Because of this lower position, ∼four upper site methylene groups and ∼ten lower site methylene groups are pulled into the central cavity leaving a smaller portion of the upper and lower legs in their respective binding sites ( Fig. 5a-b , and S18b). This lower pose also reveals density contacting the C-helices that is consistent with phosphatidylethanolamine head groups ( Fig. 5a-b , S18a). The observation that the R257A affects lipid plug position indicates that this residue has a key role in lipid plug binding and suggests that the functional effects from mutations at this site ( Fig. 4c ) are rooted in alteration of lipid plug binding. Importantly, the fact that we can observe positional changes in the lipid plug provides corroborating evidence regarding its assignment and role as a crucial component of the TWIK2 structure. Download figure Open in new tab Figure 5 Comparison of plugged and unplugged K 2P 6.1 (TWIK2) R257A structures. a, Superposition of TWIK2:ND (deep teal and bright orange) and TWIK2 RM R257A:Det (plugged) (deep blue) structures. b, Close up view of the central cavity from ‘a’. Lipid plugs from TWIK2:ND and TWIK2 RM R257A:Det are shown as spheres (grey) and sticks (deep blue), respectively. d, Superposition of TWIK2:ND (deep teal and bright orange) and TWIK2 RM R257A:Det (unplugged) (dirtyviolet) structures. d, Close up view of the central cavity from ‘c’. Lipid plug from TWIK2:ND is shown as spheres (grey). Discussion A puzzling aspect of many ion channel structures has been the identification of fenestrations from the central cavity that contain lipid or lipid-like densities that could represent elements that interfere with the passage of ions through the channel central cavity 4 , 6 , 7 , 44 . In particular, this phenomenon has been observed for a number of K 2P s, including K 2P 1.1 (TWIK1) 4 , 5 , K 2P 4.1 TRAAK 6 , 7 , K 2P 2.1 (TREK1) 12 , and K 2P 10.1 TREK2 8 . However, in each case the reported densities only define a single alkyl chain 4 , 6 leaving open the possibility that its origin could be detergents required for channel purification rather than a lipid 4 , 12 . One interpretation of this density is that it represents a mechanism in which horizontal lipid access from the bilayer acts as a gating mechanism 5 , 6 ; however, this issue remains controversial 13 , 27 , 45 , particularly as simulations indicate that even though lipid tails can enter the cavity from the bilayer they do not block the pore 13 , 27 . Here, we provide evidence for TWIK2 block by a pair of two-legged lipids, the lipid plug, in which each lipid leg fills a separate binding site that extends towards the bilayer and the lipid headgroup points towards the inner cytoplasmic face in an orientation that matches that of bilayer inner leaflet lipids. The upper leg site overlaps with the position assigned as single alkyl chain in the initial structure of K 2P 1.1 (TWIK1) 4 , 5 framed by P2, M2, and M4 ( Fig. 2b ). The lower leg passes through a gap on the opposite side of the channel between M2, M4, and the C-helix. In this binding mode, each of the plug lipids spans the inner cavity. Given the high similarity between TWIK1 and TWIK2 structures (Fig. S3a-c), excellent overlap of the upper leg site ( Fig. 3b ), and presence of the lower cavity in TWIK1 13 (Fig. S8d), our data suggest that the lipid block we observe occurs in both channel types. This interpretation is strengthened by the fact that cryo-EM maps from TWIK1 in an inactive (pH 5.5) and active (pH 7.4) forms have a bifurcated density in the central cavity that is well matched by the lipid plug structure derived from our TWIK2 studies ( Figs. 2e and S8e). Thus, previous observations of density in the upper leg site may represent incomplete density for similar lipid plug structures arising from lipid dynamics. Together, these data reveal a lipid binding mode that has not been noted previously and offer a reinterpretation of previous findings regarding how lipids bind TWIK channel fenestrations. Structural analysis of the R257A mutant that removes interactions with the blocking lipids leads to a repositioning of the lipid plug and suggests an important role for the conserved Arg257 residue in TWIK2 channels. It is worth noting that TWIK1 and TWIK2 have narrower inner pores than channels from the TREK subfamily, suggesting that the lipid plug structure is a distinct feature of the TWIK subfamily. Ion concentrations inside various intracellular organelles vary and there are a growing number of ion channels whose activity affects diverse intracellular compartments 46 , including TWIK2 47 . Because every ion channel has the potential to perturb ion flux, it is essential that these protein pores open only in the right place at the right time. The majority of channels in the VGIC superfamily that are activated by voltage or ligands have an intracellular gate that blocks ion passage, rendering them inactive in the absence of a stimulus 48 . This structural barrier avoids openings in the wrong subcellular compartment. Although their activity can be tuned by various types of inputs 2 , 15 , the fundamental property of K 2P s is that they are leak channels, lacking the strongly closed state of their VGIC superfamily relatives 2 , 15 . Consequently, their basal activity gives K 2P s the capacity to influence ion flux across both internal and external membranes as they transit through various cellular compartments during their life cycle. Such basal activity also has the potential to inadvertently lead to aberrant functional effects if it is unregulated. We propose that the propensity of TWIK channels to be plugged by lipids represents a regulatory mechanism that contributes to the ability of a cell to control K 2P function ( Fig. 6 ). Such changes in activity may be linked processes in which channel activation involves redistribution of TWIK2 channels from intracellular organelles to the plasma membranes as has been suggested during macrophage inflammasome activation 30 , 32 – 34 . Together, our observations suggest the hypothesis that TWIK family activation is a ‘right place, right time’ process in which the channels remain plugged until they arrive at the cellular destination where their activity is required. Download figure Open in new tab Figure 6 Model of K 2P 6.1(TWIK2) regulation. Lipid plug prevents ion passage. Removal of plug in response to cellular localization changes, changes in the membrane lipid composition, or other factors yields channels that can conduct ions. Although our data establish that the channel can exist in both plugged and unplugged forms, how the lipid plug moves in and out is unclear. The presence of the plug is incompatible with ion movement through the central cavity ( Fig. 3h ). Hence, its removal from the cavity is essential to yield a functional channel. Changes in the positions of the M4 and the C-helix would offer a potential exit path as such motions would alter the size of the lateral gap facing the bilayer that forms the most natural way for lipid egress. Whether such conformational transformations constitute an autonomous mechanism driven by changes in lipid bilayer properties or requires the intervention of factors that facilitate lipid removal remains an open question in need of investigation. Wetting-dewetting of the narrow hydrophobic inner pore has been proposed as an important regulatory mechanism for TWIK channels that can be influenced by occupation of the lateral fenestration by lipid tails 13 , 49 , 50 . As such, there may also be some role for the competition between water and the lipid plug. Further, a recent structure of TWIK2 that was reported while this work was in preparation suggests that the density previously assigned to a single alkyl chain that we assign to the upper leg of the plug can be displaced by pimozide, a drug that modulates TWIK2, raising the possibility of competition between the lipid plug and small molecule modulators of K 2P function 51 . Besides the new insights into mechanisms of lipid modulation, the TWIK2 structure highlights the unusual divergence from the canonical selectivity filter sequences afforded by the heteromeric nature of the K 2P SF 15 , 39 . The presence of a large, non-canonical residue at Y111, a site usually occupied by smaller residues ( Fig. 1c ) shows two conformations. The ‘up’ conformation is predominant in our structures but is also accompanied by a ‘down’ conformation in the lipid nanodisc complex that matches the pose found for the equivalent position in other K 2P s (Fig. S7b-d). These ‘up’/’down’ conformations match those of a regulatory histidine, His98, in K 2P 3.1 (TASK-1) 42 , 43 (Fig. S7e), and together with the functional effects of Tyr111 mutations ( Fig. 4d-e ) highlight the role of this site in K 2P function and the importance of the K 2P C-type selectivity filter gate 22 , 23 , 26 , 52 for TWIK2 function. The observation that the ‘up’/’down’ rotamer change can occur in different K 2P subfamilies together with additional conformational changes reported for the equivalent SF1 position in K 2P 1.1 (TWIK1) 5 (Fig. S7f) and K 2P 2.1(TREK-1) 25 under conditions associated with inactivation of the channel C-type SF gate highlight the functional importance of structural changes at this site. Structures ofTWIK2 in lipid and detergent environments shows that this channel conforms to the overall architecture found throughout the K 2P family and is most similar to the other structurally characterized member of the TWIK subfamily, TWIK1. Nevertheless, the TWIK2 structure revealed three new elements: an extra helix in the extracellular cap domain, the ear helix, that connects the cap with the loop leading to the first pore helix (P1), conformational variability at a position in SF1 that is linked to C-type gating in other K 2P s 5 , 25 , 42 , 43 , and the lipid plug. Understanding the roles of these elements in K 2P 6.1 (TWIK2) function will be important for dissecting the biological functions of this channel including how it affects macrophage NLRP3 inflammasome activation 30 , 32 – 34 . Our reinterpretation of how lipids affect TWIK channels by acting as a plug that binds to two lateral gaps in the channel pore sets a new framework for addressing how ion channel-lipid interactions control function that may have general relevance for other ion channel classes. Author Contributions A.M., S.N., and D.L.M. conceived the study and designed the experiments. A.M. expressed and purified the proteins, prepared the cryo-EM samples, collected and analyzed the cryo-EM data, built and refined the models. S.N. collected and analyzed the two-electrode voltage clamp data. A.M., S.N., and D.L.M. analyzed the data. D.L.M. provided guidance and support. A.M., S.N., and D.L.M. wrote the paper. Competing interests The authors declare no competing interests. Materials and Methods References Materials and Methods Expression and purification of K2P6.1WT and mutants The gene for a construct of the human K 2P 6.1 (TWIK-2) (Uniprot ID: Q9Y257), K 2P 6.1 WT , spanning residues 1-313 followed by a 3C protease cleavage site, monomeric enhanced green fluorescent protein (mEGFP), and a His 8 tag was cloned into a modified pFastBac expression vector in which the polyhedrin promotor was replaced by a mammalian cell active CMV promotor 54 . Expression vectors for TWIK-2 ERM (I289A, L290A and Y308A) and TWIK-2 ERM R257A mutants were made on this background. These vectors were used for recombinant bacmid DNA generation using chemically competent DH10EmBacY (Geneva Biotech) cells. These bacmids were then used to transfect Spodopetera frugipdera (SF9) cells to make baculoviruses for the constructs of interest 55 . Expi293F cells (Gibco A14528) were grown to 2.5–3 x 10 6 cells mL -1 at 37 °C supplemented with 8 % CO 2 shaking at 120 RPM before transduction with 10 % (v/v) baculovirus stock of K 2P 6.1 WT or mutants. 12 – 14 h after transduction, 10 mM sodium butyrate was added to the culture to enhance protein expression 56 . Flasks were then moved to 30 °C for 48h before harvesting the cells using centrifugation at 2300 g for 20 min. The pellet was gently washed with Dulbecco’s phosphate buffered saline (Gibco 14190144) to remove leftover culture media, centrifuged (3500 x g for 20 min) to recover the cell pellet, and then flash frozen with liquid nitrogen for storage at – 80°C. The pellet from 1.8 L culture was resuspended in 50 mL hypotonic buffer (20 mM KCl, 10 mM Tris-HCl pH 8.0, phenylmethylsulfonyl fluoride (PMSF), 0.1 mg mL -1 DNase1, supplemented with Pierce protease inhibitor (Thermo Fischer Scientific) and was gently stirred on a Mono Direct Variomag magnetic stirrer (Thermo Fischer Scientific) at 4 °C for 30 min. The membranes were isolated by ultracentrifugation at 185,500 g for 30 mins. Using a 16 G Precision Glide needle and 30 mL syringe, the membrane pellet was homogenized in 200 mL solubilization buffer (buffer S) (100 mM KCl, 50 mM Tris-HCl pH 8.0, supplemented with 1 mM PMSF, 0.1 mg mL -1 DNase1, 1 % (w/v) n-Dodecyl-β-D-Maltopyranoside (β-DDM), and Pierce protease inhibitor. Membrane solubilization and protein extraction was performed by gently stirring at 4°C for 3 h followed by ultracentrifugation at 185,500 g for 40 min to remove cell debris. 5ml of anti-GFP nanobody conjugated resin conjugated CNBr Sepharose beads (GE Healthcare, #17-0430-02) 57 pre-equilibrated with 200 mM KCl, 0.01% β-DDM, 20 mM Tris-HCl pH 8.0 buffer (buffer C) was added to the supernatant after the ultracentrifugation and incubated at 4°C with gentle rotation on a Boekel 260200 Orbiton platform rotator. Subsequently, the resin was collected on a gravity flow column and washed with 10 column volumes (CV) of buffer A (200 mM KCl, 20 mM Tris-HCl pH 8.0, 0.1% β-DDM), followed by another wash step with 10 CV of buffer B (200 mM KCl, 20 mM Tris-HCl pH 8.0, 0.05% β-DDM), and then 10 CV of buffer C (200 mM KCl, 20 mM Tris-HCl pH 8.0, 0.01% β-DDM). On column cleavage of the affinity tag was achieved by incubating the resin overnight at 4 °C without any shaking with buffer C supplemented to contain 200 mM KCl, 1 mM EDTA, and 3C protease at ratio of 50:1 resin volume:protease volume. The cleaved sample was collected in 50 mL falcon tube, and the resin was subsequently washed with 2 column volumes (CV) of buffer C. The wash was also collected in the same 50 mL falcon tube. This purified sample was concentrated using Amicon Ultra-15 centrifugal unit with 100 kDa cutoff and applied to a Superdex 200 increase column pre-equilibrated with buffer C. Peak fractions were analyzed by SDS-PAGE and concentrated to 2 mg mL -1 for cryo-EM sample preparation using an Amicon Ultra-0.5 ml centrifugal unit with 100 kDa cutoff. For nanodisc (ND) reconstitution, Brain Extract Polar lipid (Avanti polar lipids #141101P) was prepared by dissolving in 100 mg of lipid in 0.2 mL chloroform, drying under nitrogen gas, and followed by lyophilization. The lyophilized lipid was dissolved by sonication in 1mL of lipid buffer (200 mM KCl, 50 mM Tris pH 8.0, 0.5 % β-DDM). TWIK2 from the size-exclusion chromatography step, purified MSP1E1 38 , and prepared lipid stock were incubated together at 1:5:250 molar ratio (nanodisc reconstitution mix) for 1 h in a 1.5mL Eppendorf microcentrifuge tube at 4 °C. Bio-beads SM-2 adsorbents (Bio-rad #1528920) were prepared by washing 200 mg bio-beads with 1mL methanol, followed by 1mL water and then with 1mL buffer N (200 mM KCl, 20 mM HEPES pH 7.4). 100 mg of bio-beads equilibrated with buffer N were added to 1 mL nanodisc reconstitution mix and incubated for 1 h gently rotating at 4 °C. After 1hr the reconstitution mix was transferred to a clean 1.5 mL Eppendorf tube and incubated with another 100 mg of equilibrated bio-beads and was rotated gently at 4 °C overnight. The supernatant from the reaction mixture was injected onto Superose 6 increase 10/300 column pre-equilibrated with buffer N. Peaks were analyzed by SDS-PAGE and concentrated to 3 mg mL -1 for cryo-EM sample preparation using an Amicon ultra-0.5 mL centrifugal unit with 100 kDa cutoff. Twik-2 RM and Twik-2 RM R257A were expressed and purified by following identical methods as used for Twik-2 WT . Following the final Superose 6 Increase 10/300 GL purification step, purified samples were concentrated using Amicon ultra-0.5 mL centrifugal units to 3 mg mL -1 for cryo-EM sample preparation. Cryo-EM sample preparation and Data collection 3.5 µL of concentrated protein sample was applied to a glow-discharged (30 s at 15 mA) grid (Quantifoil Au R1.2/1.3 300 mesh) and after a wait time of 5 s, grids were blotted once for 2 – 6 s (4 °C and 100 % humidity) using a FEI Vitrobot Mark IV (Thermo Fisher Scientific) and plunge-frozen in liquid ethane. The grids were screened using Serial EM 58 on 200 kV Talos Arctica cryo-TEM (Thermo Fisher Scientific) equipped with K3 direct detector camera (Gatan) at the University of California, San Francisco (UCSF) EM facility. Movies were collected using Serial EM 58 and EPU (Thermo Fisher Scientific, https://www.thermofisher.com/us/en/home/electron-microscopy/products/software-em-3d-vis/epu-software.html ) data acquisition software on 300 keV FEI Titan Krios (Thermo Fisher Scientific) at UCSF and SLAC National Accelerator Laboratory, respectively. Both microscopes were equipped with a K3 direct-electron detector and post-BioQuantum GIF energy filter (Gatan), set to a slit width of 20 eV. Super-resolution counting mode was used at a nominal magnification of x105,000 with a pixel size of 0.43 Å (SLAC) and 0.42 Å (UCSF). A nominal defocus range between -1.1 to -2.5 µm was used with total dose of 60 e - / Å 2 , having total exposure time of 2.8 s at SLAC and -1.1 to -2.2 µm defocus range was used with total dose of 47.7 e - / Å 2 , having total exposure time 2 s at UCSF EM facility. Structure determination (image processing, model building and refinement) A total of 10,315, 13,561, 11,690 and 24,106 movies were collected for TWIK2:ND, TWIK2:Det, TWIK2 RM :Det and TWIK2 RM R257A:Det samples, respectively. Cryo-EM data processing was performed using cryoSPARC (version 4.5) 59 and Relion (v5) 60 . Patch motion correction (2x binned) and patch CTF estimation were performed before manually curating the exposures by ice-thickness, CTF-fit resolution. Selected micrographs were used for reference-free circular blob picker (diameter 60-120 Å) for particle picking followed by extraction at a box size of 260 Å. Several rounds of 2D classification were performed and further cleaning was done by categorizing particles by 3D Ab initio reconstruction. Finally, non-uniform and local refinement were performed to improve resolution and map quality. Datasets corresponding to Twik-2 RM and Twik-2 RM R257A yielded plugged conformations after non-uniform refinement. The refined particle sets for each of these datasets were subjected to 3D classification using a focused mask corresponding to the lipid-plug density obtained in non-uniform refinement. The 3D classification segregated the total particle stacks in two major groups (plugged and un-plugged). These two groups were further subjected to local refinement to obtain the final maps corresponding to Twik-2 RM (plugged and un-plugged) and Twik-2 RM R257A (plugged and un-plugged). A similar approach, generating lipid plug mask using the density observed in the vestibule region and using it for focused 3D classification, was attempted to find un-plugged conformations in the TWIK2:ND and TWIK2:Det datasets. However, this approach did not generate any 3D class without a lipid plug for these two datasets, indicating an absence of the an unplugged class. A preliminary model based on K 2P 2.1 (TREK-1)(PDB ID: 8UE2) 61 was converted to poly-alanine and used to dock as a backbone in the density using phenix.dock_in_map (v1.21.2) 62 . Multiple rounds of iterative model building and refinement was performed using Coot (v0.9.8.95) 63 and phenix.real_space_refine 64 , respectively. Final geometry validation statistics were calculated by MolProbity 65 . Figures were prepared and model comparisons were performed using UCSF ChimeraX (version 1.15) 66 and the Pymol package ( http://www.pymol.org/pymol ). Contact analysis was performed using LIGPLOT 67 , 68 . Two-electrode voltage-clamp (TEVC) electrophysiology Xenopus laevis oocytes were harvested according to UCSF IACUC Protocol AN193390 and digested using collagenase (Worthington Biochemical Corporation, #LS004183, 0.8-1.0 mg mL -1 ) in Ca 2+ -free ND96 (96 mM NaCl, 2 mM KCl, 3.8 mM MgCl 2 , 5 mM HEPES pH 7.4). Prior to RNA injection, oocytes were maintained at 4 °C in ND96 (96 mM NaCl, 2 mM KCl, 1.8 mM CaCl 2 , 2 mM MgCl 2 , 5 mM HEPES pH 7.4) supplemented with antibiotics (100 units ml -1 penicillin, 100 µg ml -1 streptomycin, 50 µg ml -1 gentamicin) and used for experiments within one week of harvest. Human K 2P 6.1 (TWIK-2) (Uniprot ID: Q9Y257) was subcloned into a previously reported pGEMHE/pMO vector 22 . Site directed mutations were made using Inverse PCR 69 with fully overlapping primers. mRNA for oocyte injections was prepared from linearized plasmid DNA (linearized with AflII) using mMessage Machine T7 Transcription Kit (Thermo Fisher Scientific). RNA was purified using RNEasy kit (Qiagen) and stored as aliquoted stocks at -80 °C. Defolliculated stage V-VI oocytes were microinjected with 11-13 ng mRNA and incubated at 18°C for expression. Currents were recorded within 48-54 h after microinjection. For amplitude comparisons, currents were compared at the same time of expression for sample injected with equivalent amounts of mRNA. For recordings, oocytes were impaled with borosilicate recording microelectrodes (0.2-–2.0 MΩ resistance) backfilled with 3 M KCl and were subjected to constant perfusion of ND96 at a rate of 3-5 ml min −1 . Except where otherwise indicated, recording solution was 96 mM NaCl, 2 mM KCl, 1.8 mM CaCl 2 , and 1.0 mM MgCl 2 , buffered with 5 mM HEPES, pH 7.4. For each recording, control solution (ND96) was perfused over a single oocyte until the current stabilized. Currents were evoked from a -80 mV holding potential followed by a 500 ms ramp from -140 mV to +50 mV. Data were recorded using a Axoclamp 900A amplifier (Molecular Devices) controlled by pCLAMP10.7 software (Molecular Devices), and digitized at 1 kHz using Digidata 1550B digitizer (Molecular Devices). Representative traces and dose response plots were generated in GraphPad Prism 10 (GraphPad Software, Boston). Data quantification and statistical analysis The results are from 2-4 independent oocyte batches. P values for Fig. 4c are as follows: P < 0.9999 (K 2P 6.1(Twik2) vs uninjected oocytes), P < 0.0001 (TWIK-2 vs TWIK-2 RM ), P < 0.0001 (TWIK-2 RM vs TWIK-2 RM V131D), P = 0.9768 (TWIK-2 RM vs TWIK-2 RM M135A), P < 0.0001 (TWIK-2 RM vs TWIK-2 RM M135D), P < 0.0001 (TWIK-2 RM vs TWIK-2 RM R257A) and P < 0.0001 (TWIK-2 RM vs TWIK-2 RM R257E). Statistical analysis was performed using One-Way ANOVA and multiple comparisions are performed with Tukey’s test. Error bars are S.E.M.. Full detailed statistical information is available in the source data spreadsheet. P values for Fig. 4e are as follows: P < 0. 9999 (TWIK2 vs uninjected oocytes), P < 0.0001 (TWIK-2 vs TWIK-2 RM ), P = 0.0341 (TWIK-2 RM vs TWIK-2 RM Y111A), P = 0. 9999 (TWIK-2 RM vs TWIK-2 RM Y111H) and P = 0.0068 (TWIK-2 RM vs TWIK-2 RM Y111N). Statistical analysis was performed using One-Way ANOVA and multiple comparisions are performed with Tukey’s test. Error bars are S.E.M.. Full detailed statistical information is available in the source data spreadsheet. Data Availability K 2P 6.1 (TWIK2) nanodisc and detergent structures (PDB: 9OTA, EMDB-70828 and 9OTK, EMDB-70837, respectively), K 2P 6.1 (TWIK2) RM plugged and unplugged structures (PDB: 9OV0, EMDB-70885 and 9OV9, EMDB-70894, respectively), and K 2P6.1 (TWIK2) RM R257A plugged and unplugged structures (PDB:9OVD, EMDB-70898 and 9OVE, EMDB-70899, respectively). Human K 2P 6.1 (TWIK2) sequence is Uniprot Q9Y257. Data or materials will be provided on request from the corresponding author. Acknowledgements We thank A. Cassago and P. Pascual at the S2C2 Cryo-EM Center and D. Bulkley and G. Gilbert at the UCSF Cryo-EM facility for help with microscope handling and data acquisition, M. Grabe for helpful discussions, and K. Brejc and M. Grabe for comments on the manuscript. This work was supported by NIH grant R01-MH093603 to D.L.M. Funder Information Declared NIH-NIMH , R01 MH093603 References 1. ↵ Mathie , A. , Veale , E.L. , Cunningham , K.P. , Holden , R.G. , and Wright , P.D . ( 2021 ). Two-Pore Domain Potassium Channels as Drug Targets: Anesthesia and Beyond . Annu Rev Pharmacol Toxicol 61 , 401 – 420 . doi: 10.1146/annurev-pharmtox-030920-111536 . OpenUrl CrossRef PubMed 2. ↵ Feliciangeli , S. , Chatelain , F.C. , Bichet , D. , and Lesage , F . ( 2015 ). The family of K2P channels: salient structural and functional properties . J Physiol 593 , 2587 – 2603 . doi: 10.1113/jphysiol.2014.287268 . OpenUrl CrossRef PubMed 3. ↵ Enyedi , P. , and Czirjak , G . ( 2010 ). Molecular background of leak K+ currents: two-pore domain potassium channels . Physiological reviews 90 , 559 – 605 . 90/2/559 [pii] doi: 10.1152/physrev.00029.2009 . OpenUrl CrossRef PubMed Web of Science 4. ↵ Miller , A.N. , and Long , S.B . ( 2012 ). Crystal structure of the human two-pore domain potassium channel K2P1 . Science 335 , 432 – 436 . 335/6067/432 [pii] doi: 10.1126/science.1213274 . OpenUrl Abstract / FREE Full Text 5. ↵ Turney , T.S. , Li , V. , and Brohawn , S.G . ( 2022 ). Structural Basis for pH-gating of the K(+) channel TWIK1 at the selectivity filter . Nat Commun 13 , 3232 . doi: 10.1038/s41467-022-30853-z . OpenUrl CrossRef PubMed 6. ↵ Brohawn , S.G. , Campbell , E.B. , and MacKinnon , R . ( 2014 ). Physical mechanism for gating and mechanosensitivity of the human TRAAK K+ channel . Nature 516 , 126 – 130 . doi: 10.1038/nature14013 . OpenUrl CrossRef PubMed Web of Science 7. ↵ Brohawn , S.G. , del Marmol , J. , and MacKinnon , R . ( 2012 ). Crystal structure of the human K2P TRAAK, a lipid- and mechano-sensitive K+ ion channel . Science 335 , 436 – 441 . 335/6067/436 [pii] doi: 10.1126/science.1213808 . OpenUrl Abstract / FREE Full Text 8. ↵ Dong , Y.Y. , Pike , A.C. , Mackenzie , A. , McClenaghan , C. , Aryal , P. , Dong , L. , Quigley , A. , Grieben , M. , Goubin , S. , Mukhopadhyay , S. , et al. ( 2015 ). K2P channel gating mechanisms revealed by structures of TREK-2 and a complex with Prozac . Science 347 , 1256 – 1259 . doi: 10.1126/science.1261512 . OpenUrl Abstract / FREE Full Text 9. ↵ Patel , A.J. , Maingret , F. , Magnone , V. , Fosset , M. , Lazdunski , M. , and Honore , E . ( 2000 ). TWIK-2, an inactivating 2P domain K+ channel . J Biol Chem 275 , 28722 – 28730 . doi: 10.1074/jbc.M003755200 [pii]. OpenUrl Abstract / FREE Full Text 10. ↵ Pountney , D.J. , Gulkarov , I. , Vega-Saenz de Miera , E. , Holmes , D. , Saganich , M. , Rudy , B. , Artman , M. , and Coetzee , W.A. ( 1999 ). Identification and cloning of TWIK-originated similarity sequence (TOSS): a novel human 2-pore K+ channel principal subunit . FEBS Lett 450 , 191 – 196 . doi: 10.1016/s0014-5793(99)00495-0 . OpenUrl CrossRef PubMed Web of Science 11. ↵ Chavez , R.A. , Gray , A.T. , Zhao , B.B. , Kindler , C.H. , Mazurek , M.J. , Mehta , Y. , Forasayeth , J.R. , and Yost , C.S . ( 1999 ). TWIK-2, a new weak inward rectifying member of the tandem pore domain potassium channel family . J. Biol. Chem . 274 , 7887 – 7892 . OpenUrl Abstract / FREE Full Text 12. ↵ Schmidpeter , P.A.M. , Petroff , J.T ., 2nd, Khajoueinejad , L. , Wague , A. , Frankfater , C. , Cheng , W.W.L. , Nimigean , C.M. , and Riegelhaupt , P.M. ( 2023 ). Membrane phospholipids control gating of the mechanosensitive potassium leak channel TREK1 . Nat Commun 14 , 1077 . doi: 10.1038/s41467-023-36765-w . OpenUrl CrossRef PubMed 13. ↵ Aryal , P. , Abd-Wahab , F. , Bucci , G. , Sansom , M.S. , and Tucker , S.J . ( 2015 ). Influence of lipids on the hydrophobic barrier within the pore of the TWIK-1 K2P channel . Channels (Austin ) 9 , 44 – 49 . doi: 10.4161/19336950.2014.981987 . OpenUrl CrossRef PubMed 14. ↵ Bobak , N. , Feliciangeli , S. , Chen , C.C. , Ben Soussia , I. , Bittner , S. , Pagnotta , S. , Ruck , T. , Biel , M. , Wahl-Schott , C. , Grimm , C. , et al. ( 2017 ). Recombinant tandem of pore-domains in a Weakly Inward rectifying K(+) channel 2 (TWIK2) forms active lysosomal channels . Scientific reports 7 , 649 . doi: 10.1038/s41598-017-00640-8 . OpenUrl CrossRef PubMed 15. ↵ Natale , A.M. , Deal , P.E. , and Minor , D.L. , Jr . . ( 2021 ). Structural Insights into the Mechanisms and Pharmacology of K2P Potassium Channels . Journal of molecular biology , 166995 . doi: 10.1016/j.jmb.2021.166995 . OpenUrl CrossRef 16. ↵ Lolicato , M. , Arrigoni , C. , Mori , T. , Sekioka , Y. , Bryant , C. , Clark , K.A. , and Minor , D.L. , Jr . . ( 2017 ). K2P2.1 (TREK-1)-activator complexes reveal a cryptic selectivity filter binding site . Nature 547 , 364 – 368 . doi: 10.1038/nature22988 . OpenUrl CrossRef PubMed 17. ↵ Brohawn , S.G. , Campbell , E.B. , and MacKinnon , R . ( 2013 ). Domain-swapped chain connectivity and gated membrane access in a Fab-mediated crystal of the human TRAAK K+ channel . Proc Natl Acad Sci U S A 110 , 2129 – 2134 . doi: 10.1073/pnas.1218950110 . OpenUrl Abstract / FREE Full Text 18. ↵ Li , B.B. , Rietmeijer , R.A. , and Brohawn , S.G . ( 2020 ). Structural basis for pH gating of the two-pore domain K(+)channel TASK2 . Nature 586 , 457 – 462 . doi: 10.1038/s41586-020-2770-2 . OpenUrl CrossRef PubMed 19. ↵ Rödström , K.E.J. , Kiper , A.K. , Zhang , W. , Rinne , S. , Pike , A.C.W. , Goldstein , M. , Conrad , L.J. , Delbeck , M. , Hahn , M.G. , Meier , H. , et al. ( 2020 ). A lower X-gate in TASK channels traps inhibitors within the vestibule . Nature 582 , 443 – 447 . doi: 10.1038/s41586-020-2250-8 . OpenUrl CrossRef PubMed 20. ↵ Rodstrom , K.E.J. , Eymsh , B. , Proks , P. , Hayre , M.S. , Cordeiro , S. , Mendez-Otalvaro , E. , Madry , C. , Rowland , A. , Kopec , W. , Newstead , S. , et al. ( 2025 ). Cryo-EM structure of the human THIK-1 K2P K(+) channel reveals a lower Y gate regulated by lipids and anesthetics . Nat Struct Mol Biol . doi: 10.1038/s41594-025-01497-6 . OpenUrl CrossRef 21. ↵ Roy-Chowdhury , S. , Jang , S. , Abderemane-Ali , F. , Naughton , F. , Grabe , M. , and Minor , D.L. , Jr . . ( 2025 ). Structure of the human K(2P)13.1 channel reveals a hydrophilic pore restriction and lipid cofactor site . Nat Struct Mol Biol . doi: 10.1038/s41594-024-01476-3 . OpenUrl CrossRef 22. ↵ Bagriantsev , S.N. , Peyronnet , R. , Clark , K.A. , Honore , E. , and Minor , D.L. , Jr . . ( 2011 ). Multiple modalities converge on a common gate to control K2P channel function . EMBO J 30 , 3594 – 3606 . emboj2011230 [pii] doi: 10.1038/emboj.2011.230 . OpenUrl Abstract / FREE Full Text 23. ↵ Piechotta , P.L. , Rapedius , M. , Stansfeld , P.J. , Bollepalli , M.K. , Ehrlich , G. , Andres-Enguix , I. , Fritzenschaft , H. , Decher , N. , Sansom , M.S. , Tucker , S.J. , and Baukrowitz , T . ( 2011 ). The pore structure and gating mechanism of K2P channels . EMBO J 30 , 3607 – 3619 . emboj2011268 [pii] doi: 10.1038/emboj.2011.268 . OpenUrl CrossRef PubMed Web of Science 24. Cohen , A. , Ben-Abu , Y. , Hen , S. , and Zilberberg , N . ( 2008 ). A novel mechanism for human K2P2.1 channel gating. Facilitation of C-type gating by protonation of extracellular histidine residues . J Biol Chem 283 , 19448 – 19455 . M801273200 [pii] doi: 10.1074/jbc.M801273200 . OpenUrl Abstract / FREE Full Text 25. ↵ Lolicato , M. , Natale , A.M. , Abderemane-Ali , F. , Crottes , D. , Capponi , S. , Duman , R. , Wagner , A. , Rosenberg , J.M. , Grabe , M. , and Minor , D.L. , Jr . . ( 2020 ). K2P channel C-type gating involves asymmetric selectivity filter order-disorder transitions . Science Advances 6 , eabc9174. doi: 10.1126/sciadv.abc9174 . OpenUrl FREE Full Text 26. ↵ Schewe , M. , Nematian-Ardestani , E. , Sun , H. , Musinszki , M. , Cordeiro , S. , Bucci , G. , de Groot , B.L. , Tucker , S.J. , Rapedius , M. , and Baukrowitz , T. ( 2016 ). A Non-canonical Voltage-Sensing Mechanism Controls Gating in K2P K(+) Channels . Cell 164 , 937 – 949 . doi: 10.1016/j.cell.2016.02.002 . OpenUrl CrossRef PubMed 27. ↵ Aryal , P. , Jarerattanachat , V. , Clausen , M.V. , Schewe , M. , McClenaghan , C. , Argent , L. , Conrad , L.J. , Dong , Y.Y. , Pike , A.C.W. , Carpenter , E.P. , et al. ( 2017 ). Bilayer-Mediated Structural Transitions Control Mechanosensitivity of the TREK-2 K2P Channel . Structure 25 , 708 – 718 e702. doi: 10.1016/j.str.2017.03.006 . OpenUrl CrossRef 28. ↵ Lloyd , E.E. , Crossland , R.F. , Phillips , S.C. , Marrelli , S.P. , Reddy , A.K. , Taffet , G.E. , Hartley , C.J. , and Bryan , R.M. , Jr . . ( 2011 ). Disruption of K(2P)6.1 produces vascular dysfunction and hypertension in mice . Hypertension 58 , 672 – 678 . doi: 10.1161/HYPERTENSIONAHA.111.175349 . OpenUrl CrossRef 29. ↵ Pandit , L.M. , Lloyd , E.E. , Reynolds , J.O. , Lawrence , W.S. , Reynolds , C. , Wehrens , X.H. , and Bryan , R.M . ( 2014 ). TWIK-2 channel deficiency leads to pulmonary hypertension through a rho-kinase-mediated process . Hypertension 64 , 1260 – 1265 . doi: 10.1161/HYPERTENSIONAHA.114.03406 . OpenUrl Abstract / FREE Full Text 30. ↵ Di , A. , Xiong , S. , Ye , Z. , Malireddi , R.K.S. , Kometani , S. , Zhong , M. , Mittal , M. , Hong , Z. , Kanneganti , T.D. , Rehman , J. , and Malik , A.B . ( 2018 ). The TWIK2 Potassium Efflux Channel in Macrophages Mediates NLRP3 Inflammasome-Induced Inflammation . Immunity 49 , 56 – 65 e54. doi: 10.1016/j.immuni.2018.04.032 . OpenUrl CrossRef PubMed 31. ↵ Bichet , D. , Blin , S. , Feliciangeli , S. , Chatelain , F.C. , Bobak , N. , and Lesage , F . ( 2015 ). Silent but not dumb: how cellular trafficking and pore gating modulate expression of TWIK1 and THIK2 . Pflugers Arch 467 , 1121 – 1131 . doi: 10.1007/s00424-014-1631-y . OpenUrl CrossRef PubMed 32. ↵ Huang , L.S. , Anas , M. , Xu , J. , Zhou , B. , Toth , P.T. , Krishnan , Y. , Di , A. , and Malik , A.B . ( 2023 ). Endosomal trafficking of two-pore K(+) efflux channel TWIK2 to plasmalemma mediates NLRP3 inflammasome activation and inflammatory injury . eLife 12 . doi: 10.7554/eLife.83842 . OpenUrl CrossRef 33. ↵ Zhi , Y. , Wu , X. , Chen , Y. , Chen , X. , Chen , X. , Luo , H. , Yi , X. , Lin , X. , Ma , L. , Chen , Y. , et al. ( 2023 ). A novel TWIK2 channel inhibitor binds at the bottom of the selectivity filter and protects against LPS-induced experimental endotoxemia in vivo . Biochem Pharmacol 218 , 115894 . doi: 10.1016/j.bcp.2023.115894 . OpenUrl CrossRef PubMed 34. ↵ Wu , X.Y. , Lv , J.Y. , Zhang , S.Q. , Yi , X. , Xu , Z.W. , Zhi , Y.X. , Zhao , B.X. , Pang , J.X. , Yung , K.K.L. , Liu , S.W. , and Zhou , P.Z . ( 2022 ). ML365 inhibits TWIK2 channel to block ATP-induced NLRP3 inflammasome . Acta pharmacologica Sinica 43 , 992 – 1000 . doi: 10.1038/s41401-021-00739-9 . OpenUrl CrossRef PubMed 35. ↵ Izquierdo , P. , Attwell , D. , and Madry , C . ( 2019 ). Ion Channels and Receptors as Determinants of Microglial Function . Trends in neurosciences 42 , 278 – 292 . doi: 10.1016/j.tins.2018.12.007 . OpenUrl CrossRef 36. Madry , C. , Kyrargyri , V. , Arancibia-Carcamo , I.L. , Jolivet , R. , Kohsaka , S. , Bryan , R.M. , and Attwell , D . ( 2018 ). Microglial Ramification, Surveillance, and Interleukin-1beta Release Are Regulated by the Two-Pore Domain K(+) Channel THIK-1 . Neuron 97 , 299 – 312 e296 . doi: 10.1016/j.neuron.2017.12.002 . OpenUrl CrossRef PubMed 37. ↵ Drinkall , S. , Lawrence , C.B. , Ossola , B. , Russell , S. , Bender , C. , Brice , N.B. , Dawson , L.A. , Harte , M. , and Brough , D . ( 2022 ). The two pore potassium channel THIK-1 regulates NLRP3 inflammasome activation . Glia 70 , 1301 – 1316 . doi: 10.1002/glia.24174 . OpenUrl CrossRef PubMed 38. ↵ Ritchie , T.K. , Grinkova , Y.V. , Bayburt , T.H. , Denisov , I.G. , Zolnerciks , J.K. , Atkins , W.M. , and Sligar , S.G . ( 2009 ). Chapter 11 - Reconstitution of membrane proteins in phospholipid bilayer nanodiscs . Methods Enzymol 464 , 211 – 231 . doi: 10.1016/S0076-6879(09)64011-8 . OpenUrl CrossRef PubMed Web of Science 39. ↵ Pope , L. , Lolicato , M. , and Minor , D.L. , Jr . . ( 2020 ). Polynuclear Ruthenium Amines Inhibit K2P Channels via a “Finger in the Dam” Mechanism . Cell Chem Biol 27 , 511 – 524 .e514. doi: 10.1016/j.chembiol.2020.01.011 . OpenUrl CrossRef 40. ↵ Heginbotham , L. , Lu , Z. , Abramson , T. , and MacKinnon , R . ( 1994 ). Mutations in the K+ channel signature sequence . Biophys J 66 , 1061 – 1067 . OpenUrl CrossRef PubMed Web of Science 41. ↵ Cordero-Morales , J.F. , Cuello , L.G. , Zhao , Y. , Jogini , V. , Cortes , D.M. , Roux , B. , and Perozo , E . ( 2006 ). Molecular determinants of gating at the potassium-channel selectivity filter . Nat Struct Mol Biol 13 , 311 – 318 . nsmb1069 [pii] doi: 10.1038/nsmb1069 . OpenUrl CrossRef PubMed Web of Science 42. ↵ Hall , P.R. , Jouen-Tachoire , T. , Schewe , M. , Proks , P. , Baukrowitz , T. , Carpenter , E.P. , Newstead , S. , Rodstrom , K.E.J. , and Tucker , S.J . ( 2025 ). Structures of TASK-1 and TASK-3 K2P channels provide insight into their gating and dysfunction in disease . Structure 33 , 115 – 122 e114. doi: 10.1016/j.str.2024.11.005 . OpenUrl CrossRef 43. ↵ Rödström , K.E.J. , Kiper , A.K. , Zhang , W. , Rinné , S. , Pike , A.C.W. , Goldstein , M. , Conrad , L. , Delbeck , M. , Hahn , M. , Meier , H. , et al. ( 2019 ). A unique lower X-gate in TASK channels traps inhibitors within the vestibule . bioRxiv , 706168 . doi: 10.1101/706168 . OpenUrl Abstract / FREE Full Text 44. ↵ Chen , Z. , Mondal , A. , Ali , F.A. , Jang , S. , Niranjan , S. , Montano , J.L. , Zaro , B.W. , and Minor , D.L. , Jr . . ( 2023 ). EMC chaperone-Ca(V) structure reveals an ion channel assembly intermediate . Nature 619 , 410 – 419 . doi: 10.1038/s41586-023-06175-5 . OpenUrl CrossRef PubMed 45. ↵ McClenaghan , C. , Schewe , M. , Aryal , P. , Carpenter , E.P. , Baukrowitz , T. , and Tucker , S.J . ( 2016 ). Polymodal activation of the TREK-2 K2P channel produces structurally distinct open states . J Gen Physiol 147 , 497 – 505 . doi: 10.1085/jgp.201611601 . OpenUrl Abstract / FREE Full Text 46. ↵ Hu , M. , Feng , X. , Liu , Q. , Liu , S. , Huang , F. , and Xu , H . ( 2024 ). The ion channels of endomembranes . Physiological reviews 104 , 1335 – 1385 . doi: 10.1152/physrev.00025.2023 . OpenUrl CrossRef 47. ↵ Anees , P. , Saminathan , A. , Rozmus , E.R. , Di , A. , Malik , A.B. , Delisle , B.P. , and Krishnan , Y . ( 2024 ). Detecting organelle-specific activity of potassium channels with a DNA nanodevice . Nat Biotechnol 42 , 1065 – 1074 . doi: 10.1038/s41587-023-01928-z . OpenUrl CrossRef PubMed 48. ↵ Catterall , W.A. , Wisedchaisri , G. , and Zheng , N . ( 2017 ). The chemical basis for electrical signaling . Nature chemical biology 13 , 455 – 463 . doi: 10.1038/Nchembio.2353 . OpenUrl CrossRef 49. ↵ Aryal , P. , Abd-Wahab , F. , Bucci , G. , Sansom , M.S. , and Tucker , S.J . ( 2014 ). A hydrophobic barrier deep within the inner pore of the TWIK-1 K2P potassium channel . Nat Commun 5 , 4377 . doi: 10.1038/ncomms5377 . OpenUrl CrossRef PubMed 50. ↵ Aryal , P. , Sansom , M.S. , and Tucker , S.J . ( 2015 ). Hydrophobic gating in ion channels . Journal of molecular biology 427 , 121 – 130 . doi: 10.1016/j.jmb.2014.07.030 . OpenUrl CrossRef PubMed 51. ↵ Khanra , N.K. , Wang , C. , Delgado , B.D. , and Long , S.B . ( 2025 ). Structure of the human TWIK-2 potassium channel and its inhibition by pimozide . Proc Natl Acad Sci U S A 122 , e2425709122 . doi: 10.1073/pnas.2425709122 . OpenUrl CrossRef 52. ↵ Bagriantsev , S.N. , Clark , K.A. , and Minor , D.L. , Jr . . ( 2012 ). Metabolic and thermal stimuli control K(2P)2.1 (TREK-1) through modular sensory and gating domains . EMBO J 31 , 3297 – 3308 . doi: 10.1038/emboj.2012.171 . OpenUrl CrossRef PubMed Web of Science 53. ↵ Smart , O.S. , Neduvelil , J.G. , Wang , X. , Wallace , B.A. , and Sansom , M.S . ( 1996 ). HOLE: a program for the analysis of the pore dimensions of ion channel structural models . J Mol Graph 14 , 354 – 360 , 376. doi: 10.1016/s0263-7855(97)00009-x . OpenUrl CrossRef PubMed Web of Science 54. ↵ Liao , M. , Cao , E. , Julius , D. , and Cheng , Y . ( 2013 ). Structure of the TRPV1 ion channel determined by electron cryo-microscopy . Nature 504 , 107 – 112 . doi: 10.1038/nature12822 . OpenUrl CrossRef PubMed Web of Science 55. ↵ Goehring , A. , Lee , C.H. , Wang , K.H. , Michel , J.C. , Claxton , D.P. , Baconguis , I. , Althoff , T. , Fischer , S. , Garcia , K.C. , and Gouaux , E . ( 2014 ). Screening and large-scale expression of membrane proteins in mammalian cells for structural studies . Nat Protoc 9 , 2574 – 2585 . doi: 10.1038/nprot.2014.173 . OpenUrl CrossRef PubMed 56. ↵ Morales-Perez , C.L. , Noviello , C.M. , and Hibbs , R.E . ( 2016 ). Manipulation of Subunit Stoichiometry in Heteromeric Membrane Proteins . Structure 24 , 797 – 805 . doi: 10.1016/j.str.2016.03.004 . OpenUrl CrossRef PubMed 57. ↵ Lee , H. , Lolicato , M. , Arrigoni , C. , and Minor , D.L. , Jr . . ( 2021 ). Production of K(2P)2.1 (TREK-1) for structural studies . Methods Enzymol 653 , 151 – 188 . doi: 10.1016/bs.mie.2021.02.013 . OpenUrl CrossRef 58. ↵ Mastronarde , D.N . ( 2005 ). Automated electron microscope tomography using robust prediction of specimen movements . J Struct Biol 152 , 36 – 51 . doi: 10.1016/j.jsb.2005.07.007 . OpenUrl CrossRef PubMed Web of Science 59. ↵ Punjani , A. , Rubinstein , J.L. , Fleet , D.J. , and Brubaker , M.A . ( 2017 ). cryoSPARC: algorithms for rapid unsupervised cryo-EM structure determination . Nature methods 14 , 290 – 296 . doi: 10.1038/nmeth.4169 . OpenUrl CrossRef PubMed 60. ↵ Scheres , S.H . ( 2012 ). RELION: implementation of a Bayesian approach to cryo-EM structure determination . J Struct Biol 180 , 519 – 530 . doi: 10.1016/j.jsb.2012.09.006 . OpenUrl CrossRef PubMed 61. ↵ Deal , P.E. , Lee , H. , Mondal , A. , Lolicato , M. , Mendonca , P.R.F. , Black , H. , Jang , S. , El-Hilali , X. , Bryant , C. , Isacoff , E.Y. , et al. ( 2024 ). Development of covalent chemogenetic K(2P) channel activators . Cell Chem Biol 31 , 1305 – 1323 e1309 . doi: 10.1016/j.chembiol.2024.06.006 . OpenUrl CrossRef 62. ↵ Liebschner , D. , Afonine , P.V. , Baker , M.L. , Bunkoczi , G. , Chen , V.B. , Croll , T.I. , Hintze , B. , Hung , L.W. , Jain , S. , McCoy , A.J. , et al. ( 2019 ). Macromolecular structure determination using X-rays, neutrons and electrons: recent developments in Phenix . Acta Crystallogr D Struct Biol 75 , 861 – 877 . doi: 10.1107/S2059798319011471 . OpenUrl CrossRef PubMed 63. ↵ Emsley , P. , Lohkamp , B. , Scott , W.G. , and Cowtan , K . ( 2010 ). Features and development of Coot . Acta crystallographica. Section D, Biological crystallography 66 , 486 – 501 . doi: 10.1107/S0907444910007493 . OpenUrl CrossRef PubMed Web of Science 64. ↵ Afonine , P.V. , Poon , B.K. , Read , R.J. , Sobolev , O.V. , Terwilliger , T.C. , Urzhumtsev , A. , and Adams , P.D . ( 2018 ). Real-space refinement in PHENIX for cryo-EM and crystallography . Acta Crystallogr D Struct Biol 74 , 531 – 544 . doi: 10.1107/S2059798318006551 . OpenUrl CrossRef PubMed 65. ↵ Williams , C.J. , Headd , J.J. , Moriarty , N.W. , Prisant , M.G. , Videau , L.L. , Deis , L.N. , Verma , V. , Keedy , D.A. , Hintze , B.J. , Chen , V.B. , et al. ( 2018 ). MolProbity: More and better reference data for improved all-atom structure validation . Protein Sci 27 , 293 – 315 . doi: 10.1002/pro.3330 . OpenUrl CrossRef PubMed 66. ↵ Pettersen , E.F. , Goddard , T.D. , Huang , C.C. , Meng , E.C. , Couch , G.S. , Croll , T.I. , Morris , J.H. , and Ferrin , T.E . ( 2021 ). UCSF ChimeraX: Structure visualization for researchers, educators, and developers . Protein Sci 30 , 70 – 82 . doi: 10.1002/pro.3943 . OpenUrl CrossRef PubMed 67. ↵ Laskowski , R.A. , and Swindells , M.B . ( 2011 ). LigPlot+: Multiple Ligand-Protein Interaction Diagrams for Drug Discovery . Journal of Chemical Information and Modeling 51 , 2778 – 2786 . doi: 10.1021/ci200227u . OpenUrl CrossRef PubMed 68. ↵ Wallace , A.C. , Laskowski , R.A. , and Thornton , J.M . ( 1995 ). LIGPLOT: a program to generate schematic diagrams of protein-ligand interactions . Protein engineering 8 , 127 – 134 . OpenUrl CrossRef PubMed Web of Science 69. ↵ Silva , D. , Santos , G. , Barroca , M. , and Collins , T . ( 2017 ). Inverse PCR for Point Mutation Introduction . Methods Mol Biol 1620 , 87 – 100 . doi: 10.1007/978-1-4939-7060-5_5 . OpenUrl CrossRef View the discussion thread. Back to top Previous Next Posted June 15, 2025. Download PDF Supplementary Material Email Thank you for your interest in spreading the word about bioRxiv. NOTE: Your email address is requested solely to identify you as the sender of this article. Your Email * Your Name * Send To * Enter multiple addresses on separate lines or separate them with commas. You are going to email the following A lipid plug affects K2P6.1(TWIK-2) function Message Subject (Your Name) has forwarded a page to you from bioRxiv Message Body (Your Name) thought you would like to see this page from the bioRxiv website. 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