Optimal reconstitution of ELIC in liposomes to facilitate single particle cryo-EM was achieved by dialysis. Reconstitution of ELIC in liposomes at low protein:lipid molar ratios can be accomplished by detergent removal with Biobeads [15]; however, reconstitution at a higher protein:lipid molar ratio (1:500 in this study) with efficient incorporation of ELIC in the liposomes required dialysis. The method was adapted from protocols previously reported for the reconstitution of muscle nAChR [16], 5-HT3aR [17], and ELIC [18]. Although dialysis was performed for 5-7 days to ensure complete removal of detergent, the minimum time of dialysis necessary was not tested. A shorter dialysis protocol could likely be implemented for less stable proteins depending on the detergent and dialysis conditions [19]. Proteoliposomes were formed using a 2:1:1 molar ratio of POPC (1-palmitoyl-2-oleoyl-phosphatidylcholine): POPE (1-palmitoyl-2-oleoyl-phosphatidylethanolamine): POPG (1-palmitoyl-2-oleoyl-phosphatidylglycerol); these lipids are known to support ELIC activity [15], and phosphatidylethanolamine and phosphatidylglycerol are the two principal phospholipids found in gram-negative bacteria in which ELIC is expressed. Using a stopped-flow thallium flux assay, we confirmed that WT ELIC activates and then mostly desensitizes in response to the agonist, propylamine (Fig. 1A). The time constant of activation and desensitization were 10 ± 2 ms and 5.1 ± 2.1 s (±SD, n=6), respectively, which matches the kinetics of ELIC activation and desensitization from excised patch-clamp recordings in liposomes or HEK293 cells [20–22]. We previously identified a combination of five mutations in ELIC, P254G/C300S/V261Y/G319F/I320F, called ELIC5 which eliminates channel desensitization [15]. We confirmed that ELIC5 in 2:1:1 POPC:POPE:POPG liposomes activates and does not desensitize for at least 20 minutes (Fig. 1A). The open probability of ELIC5 when activated by 10 mM propylamine in 2:1:1 POPC:POPE:POPG membranes is estimated to be close to 1 [15]. Therefore, proteoliposome preparations of WT ELIC in the absence and presence of propylamine is expected to yield resting and desensitized channels, respectively, and ELIC5 in the presence of propylamine is expected to yield activated channels. The same reconstitution protocol was used for the stopped-flow thallium flux assay and single particle cryo-EM.

We determined structures of WT ELIC with and without agonist, and ELIC5 with agonist. Side views from the 2D class averages showed that ELIC is oriented in both directions in the liposomes with a preference for the extracellular domain (ECD) facing the extra-liposomal solution (i.e. outward-facing orientation) (Fig. 1B, Supplementary Figure 1, 2 and 3). Heterogeneous refinement in CryoSPARC separated the particles into two classes that generated outward-facing and inward-facing 3D reconstructions (Fig. 1C). One caveat to this analysis is that top/down views of outward- and inward-facing channels may not be distinguishable without the curvature of the liposome membrane as a reference. Therefore, it is possible that outward- and inward-facing ELIC particles could not be rigorously separated using 3D heterogeneous refinement or 3D classification in CryoSPARC or Relion. Accordingly, the proportion of particles assigned to outward- and inward-facing 3D classes may not accurately reflect the relative orientation of ELIC in the liposomes. Nevertheless, final maps were obtained for apo WT ELIC at a resolution of 3.4 Å for outward-facing and 3.6 Å for inward-facing channels, agonist-bound WT ELIC at a resolution of 3.8 Å for outward-facing and 4.2 Å for inward-facing channels, and agonist-bound ELIC5 at a resolution of 2.8 Å for outward-facing and 4.2 Å for inward-facing channels (Fig. 2A, Supplementary Figure 1, 2, 3 and 4, and Supplementary Table 1). In all samples, the inward- and outward-facing structures of ELIC were not significantly different. However, we cannot be certain that ELIC structure is insensitive to membrane curvature due to the limitations of distinguishing inward- and outward-facing particles. In addition, the inward-facing structures were of significantly lower resolution and quality compared to the outward-facing structures. The reason for this is uncertain, but could be due to fewer inward-facing particles, increased structural heterogeneity, and contamination within the liposomes. We focused our analysis on the outward-facing structures since these have better resolution. Moreover, only outward-facing channels are activated by agonist in the stopped-flow thallium flux assay.

The structures of ELIC in liposomes have the same overall architecture as structures of ELIC or other pLGICs in nanodiscs, consisting of a homopentameric assembly around a central ion conduction pore (Fig. 2). Each subunit consists of an ECD and transmembrane domain (TMD). In the agonist-bound structures, propylamine binds in a conserved site between β-sheets from two adjacent subunits. Comparison of the apo WT ELIC and agonist-bound ELIC5 structures shows the conformational changes for activation, and comparison of agonist-bound ELIC5 and agonist-bound WT ELIC structures shows the conformation changes for desensitization. ELIC activation consists of a contraction of the agonist binding site, counter-clockwise twisting of the ECD, and outward translation of M2-M3 linker and pre-M1 linker in the TMD (Supplementary Figure 5). These changes are also associated with an outward translation and counter-clockwise twisting of the transmembrane helices (M1-M4) that leads to opening of the ion channel pore lined by the M2 helix (Supplementary Figure 5). ELIC desensitization consists of a partial return of the transmembrane helices (clockwise twisting and contraction), but without significant change in the ECD or the M2-M3 and pre-M1 linkers in the TMD (Supplementary Figure 5).

The structure of the pore also reveals the conformational state of ELIC: the presence or absence of a continuous and sufficiently wide water-accessible pathway indicates whether the ion channel is open or closed, respectively. Apo WT ELIC shows a pore with two hydrophobic constrictions at F247 and L240 (also known as 16’ and 9’ positions of M2), where the ion conduction pathway is dehydrated and therefore, closed (Fig. 2B, Supplementary Fig. 6). In contrast, the agonist-bound ELIC5 pore is hydrated and open because M2 rotates and F247 and L240 orient away from the pore axis towards the adjacent subunit (Fig. 2B, Supplementary Fig. 6). There is some narrowing of the pore at 2’ (Q233, diameter of 4.4 Å), but this hydrophilic side chain, which can take on different rotameric states [15], is not anticipated to impede ion conduction. Agonist-bound WT ELIC shows conformational heterogeneity at F247, and two states were modeled with different conformations for F247 and Y248 (Supplementary Fig. 6). In state 1, F247 orients towards the pore axis, while in state 2, F247 orients towards M2 of the adjacent subunit. The latter has a wider pore at 16’ (F247), but still 9’ (L240) forms a hydrophobic constriction (diameter of 3.3 Å) like Apo WT ELIC (diameter of 2.6 Å) (Fig. 2B, Supplementary Fig. 6). There is also a narrowing of the pore at 2’ (Q233) in the agonist-bound WT ELIC structure (diameter of 3.7 Å). Nevertheless, the pore diameter at 9’ (L240) and the hydrophobicity of this residue suggests that 9’ is the highest barrier to ion conduction in the agonist-bound WT ELIC structure. In summary, apo and agonist-bound WT ELIC are closed structures with hydrophobic constrictions at 9’ consistent with resting and desensitized states, and agonist-bound ELIC5 is an open structure consistent with an activated state. It has been proposed that the activation and desensitization gates in the GABA_AR and GlyR are distinct with 9’ being the activation gate and −2’ the desensitization gate [1, 23–26]. However, in agonist-bound structures of the α7 nAChR and 5-HT3aR, which are expected to be desensitized conformations, 9’ is the only constriction in the pore that poses an energetic barrier to ion and water permeation [4, 7, 27, 28]. It was previously suggested that the agonist-bound structures of ELIC and 5-HT3aR may be pre-active states [15, 29], and that the channel may somehow be limited from entering a desensitized state due to detergent or nanodisc conditions [12]. Based on the liposome structures, we conclude that 9’ is the desensitization gate for ELIC and possibly other cation-selective pLGICs. This conclusion is consistent with studies showing that mutation of 9’ (L240A) in ELIC appears to eliminate desensitization [30].

Lipid-like densities are also resolved in the agonist-bound ELIC5 structure (Supplementary Figure 7). The most prominent lipid densities are in the outer TMD consisting of four lipid-like tails. Two of these tails fill intersubunit (M1-M3) and intrasubunit (M3-M4) grooves, and meet to form a putative phospholipid headgroup near R117 of the β6-β7 interfacial loops. A very similar phospholipid density was observed previously in agonist-bound ELIC5 in nanodiscs (e.g. MSP1E3D1) (Supplementary Figure 7), and streamlined free energy perturbation calculations determined that this is likely POPG and not POPE or POPC [15]. Another lipid-like tail adjacent to M1 joins with the density in the intersubunit site making a three-pronged lipid-like density. The ELIC5 structure in MSP1E3D1 also showed a similar but weaker density. Therefore, the lipid densities observed in the liposome structure of the activated state of ELIC are very similar to those observed in a nanodisc structure, suggesting that ELIC and not the nanodisc are primarily determining the interaction of these phospholipids. Lipid-like densities are not well appreciated in the apo WT ELIC or agonist-bound WT ELIC structures, possibly due to the lower resolution of these structures or differences in the nature of the lipid interactions [15].

Nanodiscs affect the structure of ELIC possibly through interactions of ELIC with the membrane scaffold protein (MSP) [12, 14]. Larger nanodiscs may decrease the probability of interactions with the MSP and therefore be superior membrane mimetics [14]. To test this possibility, we reconstituted WT ELIC in spNW25, a circularized nanodisc with an average diameter of 25 nm [31], for single particle cryo-EM analysis, and determined a structure of ELIC in the presence of agonist (propylamine). spNW25 nanodiscs are significantly larger than the 11 nm (spMSP1D1) and 15 nm (spNW15) nanodiscs previously used for agonist-bound WT ELIC structures [12]. Initial inspection of the micrographs showed multiple ELIC particles in most nanodiscs, and localization of the particles to the rim of the nanodisc (Fig. 3A). Consistent with this observation, side views of the 2D class averages (Fig. 3B) and the resulting 3D reconstruction using C1 symmetry (Fig. 3C, Supplementary Figure 8) showed significant MSP density on one side of the TMD. A recent study showed that MSP densities can be resolved in the structures of many membrane proteins [32]. We therefore re-analyzed the dataset of agonist-bound WT ELIC in spMSP1D1 with C1 symmetry; the resulting map showed strong MSP densities associated with ELIC (Fig. 3E). Taken together, the structures indicate that ELIC has a strong affinity for the MSP or nanodisc rim irrespective of nanodisc size.

We next examined whether the C1 reconstructions of agonist-bound WT ELIC in spMSP1D1 or spNW25 nanodiscs exhibit any asymmetric features associated with contact with the MSP. In the spMSP1D1, the cryo-EM map showed stronger densities for M4 in subunits adjacent to the MSP, and the local resolutions of those subunits are higher in both the TMD and ECD (Fig. 3E and 3F). This agrees with the finding in other nanodisc-reconstituted membrane proteins that interactions with the MSP stabilize the local conformation of the protein [32]. In contrast, the C1 reconstruction of agonist-bound ELIC in spNW25 showed better local resolution in subunits facing the lipid bilayer (Fig. 3D). In addition, M4 was only resolved in one of the lipid bilayer-facing subunits (Fig. 3C). A C5 reconstruction of agonist-bound WT ELIC in spNW25 also did not show densities for M4 (Supplementary Figure 8). The C1 maps of agonist-bound ELIC in nanodiscs suggest that the MSP can influence the local dynamics of the protein.

The micrographs and 2D class averages of ELIC in spNW25 showed multiple ELIC particles in each nanodisc. ∼10% of particles from the 2D class averages showed two adjacent pentamers (Supplementary Figure 8), but these dimers of pentamers did not yield high resolution reconstructions. 2D class averages of dimers of pentamers were never observed in the liposome datasets. This observation suggests that reconstitution of membrane proteins in large nanodiscs may be a strategy to promote the association of multiple membrane proteins. Whether the formation of dimers of pentamers alters the function of ELIC is not known.

The structures of ELIC in nanodiscs differ from the structures in liposomes to varying degrees. From this and previous studies [12, 15], the following structures of ELIC in nanodiscs have been determined: apo WT ELIC in MSP1E3D1 (PDB 8D65) and spMSP1D1 (PDB 8F35), agonist-bound ELIC5 in MSP1E3D1 (PDB 8VUW) and spNW15 (PDB 8TWV), and agonist-bound WT ELIC in MSP1E3D1 (PDB 8D66), spMSP1D1 (PDB 8F34), spNW15 (PDB 8TWZ), and spNW25 (PDB 9NH4). To examine the effect of nanodisc size on ELIC structure, we compared agonist-bound WT ELIC in liposomes with spMSP1D1 (11 nm), spNW15 (15 nm) and spNW25 (25 nm). Unexpectedly, the structure in the largest nanodisc, spNW25, deviates most from the liposome structure. There is a shortening of M1 and M2, and tilting of M3 towards the membrane that decreases the height of these transmembrane helices along the pore axis by ∼1-1.5 Å compared to the liposome and spMSP1D1 structures (Fig. 4A, Supplementary Table 2). Along with this, M2 is tilted towards the pore axis, narrowing the pore even further at L240 (9’) and Q233 (2’) (Fig. 4B). In spNW15, M2 tilts towards the pore axis at the intracellular end slightly narrowing the pore, as was reported previously [12]. The spMSP1D1 structure most closely matches the liposome structure, except there is ∼1 Å outward (away from the pore axis) translation of M4 in the liposome structure compared to the spMSP1D1 structure (Fig. 4C). There is no M4 density in the C5 maps of agonist-bound WT ELIC in spNW15 and spNW25, suggesting that larger nanodiscs increase the structural heterogeneity of this helix.

In apo WT ELIC, the structures in MSP1E3D1 or spMS1PD1 are not significantly different from the structure in liposomes. In agonist-bound ELIC5, M4 is translated (2 Å) and tilted (∼5 degrees) outward (away from the pore axis) in the liposome structure compared to the MSP1E3D1 or spNW15 structures (Fig. 4D). Along with this, there is a slight outward expansion of the M1, M2 and M3 transmembrane helices away from the pore axis, indicating that the liposome is producing an activated structure that is more loosely packed.

We further analyzed the agonist-bound WT ELIC and ELIC5 structures by measuring the area between subunit interfaces in the TMD and the buried surface area between M4 and the rest of the protein. There is an overall trend where the liposome structures having a smaller area between subunit interfaces in the TMD and a smaller buried surface area between M4 and the rest of the protein (Supplementary Table 3). This means that the TMD is more loosely packed in the agonist-bound structures in liposomes compared to nanodiscs. Accordingly, the total buried surface area is also consistently smaller in the liposome structures compared to the nanodisc structures (Supplementary Table 3).

In summary, the agonist-bound structures of WT ELIC or ELIC5 in liposomes deviate from the nanodisc structures, particularly in M4. The structure of the TMD in liposomes is generally more loosely packed than in nanodiscs. This is consistent with MD simulations of ELIC in nanodiscs showing that the outward tilt of M4 is reduced by interactions with the MSP [12]. Contrary to what was previously suggested [12], the agonist-bound WT ELIC structure in a large nanodisc (spNW25) deviates most from the structure in liposomes indicating that such large nanodiscs do not recapitulate the membrane environment of a liposome.

ELIC was expressed from pET-26-MBP-ELIC provided by Raimund Dutzler (Addgene plasmid #39239) in OverExpress C43 (DE3) E. coli (Lucigen 60446-1) as previously described [40]. The ELIC5 mutant (P254G/C300S/V251Y/G319F/I320F) was made using Quikchange and confirmed by Sanger sequencing (Genewiz). Cultures were grown with Terrific Broth (Sigma T0918) using 0.1 mM IPTG (Sigma I6758) for induction. The cells were lysed with an Avestin C5 emulsifier, and membranes solubilized with 1% DDM (Anatrace D310S). The extracted protein was purified with amylose resin (New England Biolabs E8022L), and eluted with 40 mM maltose in buffer A (10 MM Tris pH 7.5, 100 mM NaCl, 0.02% DDM). The maltose binding protein was removed by overnight digestion with HRV-3c protease (ThermoFisher Scientific 88947). Lastly, the protein was purified over a Sephadex 200 Increase 10/300 size exclusion column (Cytiva 28-9909-44) in Buffer A.

Reconstitution of WT ELIC or ELIC5 in liposomes was performed using dialysis. 2:1:1 POPC:POPE:POPG was dried overnight in a vacuum desiccator to remove chloroform, resuspended in 50 mM HEPES pH 7, 150 mM NaCl, 2% cholate at 10 mg/ml, and sonicated to clarity. The lipid solution was diluted to 5 mg/ml to 1% cholate. For each sample, 0.5 mg of lipid (100 μl) was mixed with 250 μg of WT ELIC or ELIC5 at ∼7 mg/ml, producing a ELIC(pentamer):lipid molar ratio of ∼1:500. For samples with agonist, propylamine was added at this step to a final concentration of 10 mM such that the proteoliposomes will have propylamine in both the extraliposomal and intraliposomal space. The sample was incubated on ice for 1h, and then dialyzed (ThermoScientific 88402-20K MWCO) against 50 mM HEPES pH 7, 150 mM NaCl with or without agonist over 5-7 days with 6x solution changes of 14 ml each. After dialysis, the sample was ∼150 μl. The sample was centrifuged at 10,000xg for 5 min to remove large aggregates, and concentrated to ∼50-75 μl using a Amicon Ultra-0.5 100 kDa MWCO centrifugal filter (Millipore Sigma UFC910024). The final lipid concentration was ∼5 mg/ml, and this was used directly for freezing for cryo-EM analysis.

spNW25 was expressed and purified from Addgene plasmid #173484 provided by Huan Bao exactly as previously described using Ni-NTA affinity purification, but without size exclusion chromatography [31]. Reconstitution of WT ELIC in spNW25 was performed using a liposome destabilization technique as described previously [15]. 2:1:1 POPC:POPE:POPG in 50 mM HEPES pH 7, 150 mM NaCl at 7.5 mg/ml was freeze-thawed 3x and extruded with a 400 nm filter (Avanti Polar Lipids). These lipids were destabilized by adding 0.4% DDM and incubating at RT for 3 h. ELIC and spNW25 was added to the sample with a molar ratio of 1:2:600 of ELIC:spNW25:lipid and incubated at RT for 1.5 h. DDM was removed to form nanodiscs with Biobeads SM-2 Resin (Bio-Rad 1528920) over night at 4 °C. Nanodiscs were further purified over a Sephadex 200 Increase 10/300 column in 50 mM HEPES pH 7 with 150 mM NaCl. The nanodisc fractions were concentrated to ∼0.5 mg/ml, and propylamine was added to a final concentration of 50 mM at least 30 min prior to freezing for cryo-EM analysis.

Cryo-EM samples for agonist-bound WT ELIC in spNW25 were prepared as previously described [12]. 3 μl of sample was applied to Quantifoil R2/2 copper grids, previously negatively glow discharged using a GloQube (Quorum Technologies) for 60 s. The grids were blotted for 2 s in a 95% humidity environment and vitrified in liquid ethane with a Vitrobot Mark IV (ThermoFisher Scientific). Images were collected on a Glacios 200 kV Cryo-EM equipped with a Falcon 4 Direct Electron Detector (ThermoFisher Scientific). The single particle cryo-EM data was acquired by counting mode on the Falcon 4 with EPU software (version 3.5.1). The movies were collected with a pixel size of 1.184, and defocus range of −0.8 to −2.4 μm. Each movie consisted of 45 individual frames with a total dose of 46.6 electrons per Ų.

For the proteoliposome samples, 3 μl of sample was applied to a Quantifoil R2/2 gold grid and incubated for 3 min in a 95% humidity environment. The grid was then blotted for 2 s, followed by a second sample application. After the second sample application, the grid was incubated at 95% humidity for 30 s, blotted for 2 s, and vitrified in liquid ethane. Images were collected on a Krios 300 kV Cryo-EM equipped with a Falcon 4 Direct Electron Detector. Single particle cryo-EM data was acquired using counting mode on the Falcon 4 with the EPU software 3.8.1. For apo WT ELIC and agonist-bound ELIC5, movies were collected with a pixel size of 0.868, and for agonist-bound WT ELIC, movies were collected with a pixel size of 0.865. A total dose of 55.4 electrons per Ų was used for apo WT ELIC, 54.4 electrons per Ų for agonist-bound WT ELIC, and 56.9 electrons per Ų for agonist-bound ELIC5.

Single particle cryo-EM analysis of WT ELIC in spNW25 with 50 mM propylamine was performed using CryoSPARC v4.6.0 [41] and Relion5.0. Initial processing was performed with CryoSPARC including Patch Motion Correction, Patch CTF and particle picking using templates from a previously determined ELIC map. Extracted particles were cleaned up with multiple rounds of 2D classification. To increase the number of picked particles, Topaz particle picking was performed by separately training with top/down, tilted and side views of ELIC from the last 2D classification job, and these particles underwent multiple rounds of 2D classification. The resulting ∼787,500 particles underwent multiple heterogeneous refinement jobs using C1 symmetry. For the C1 reconstruction, the best class from the final heterogeneous refinement job was further analyzed in Relion5.0 using multiple rounds of 3D classification with blush regularization and C1 symmetry. The final post-processed map was generated in Relion5.0. For the C5 reconstruction, the ∼787,500 particles underwent multiple heterogeneous refinement jobs in CryoSPARC using C5 symmetry, followed by multiple 3D classifications in Relion5.0 with blush regularization and C5 symmetry. The final post-processed map was generated in Relion5.0.

Each of the three proteoliposome samples were analyzed in a similar fashion as the approach used for the ELIC spNW25 structure, and the workflow is highlighted in Supplementary Figures 1-3. Movies were motion corrected and CTF estimation was performed in cryoSPARC. Particle picking was performed with blob picker, which yielded poor results, but many rounds of 2D classification eventually yielded a small number of particles (several thousand) producing class averages that resembled top/down, tilted and membrane-embedded side views. These particles were used for Topaz training and the extracted particles were further processed by 2D classification. The resulting 2D class averages were improved and, again, used for Topaz training separating top/down and tilted from side views. This was performed 2-3 times for each dataset. When there was no significant increase in the number of good particles from Topaz picking, an ab-initio reconstruction was performed to generate initial volumes for heterogeneous refinement. It was apparent from the side views of the 2D class averages that ELIC was oriented inward and outward in the liposomes. Accordingly, ab-initio reconstructions produced volumes with opposite membrane curvature indicative of inward- and outward-facing channels. Multiple rounds of heterogeneous refinement with C1 and C5 symmetry were performed with the inward- and outward-facing volumes as well as 2-3 junk volumes. The C1 refinement jobs did not produce 3D reconstructions of sufficient quality for further refinement. Therefore, we proceeded with refinement and 3D classification using C5 symmetry. The resulting particles were used for non-uniform refinement and local refinement with a mask for the entire ELIC or ELIC5 protein. The local refinement job generally produced better maps probably because it masked out the liposome membrane from the refinement procedure. In general, better maps could be obtained with further processing in Relion5.0, except for agonist-bound ELIC5 which yielded a final outward-facing map of 2.8 Å resolution. Particles from inward- and outward-facing classes underwent multiple rounds of 3D classification in Relion5.0 without and with a mask for the entire ELIC protein. The masked 3D classification jobs yielded the best final classes for post-process refinement.

For the apo WT ELIC structures in liposomes, an initial model of ELIC was obtained from PDB 8F35. For all agonist-bound WT structures, an initial model was obtained from PDB 8F34. For the agonist-bound ELIC5 structures in liposomes, an initial model was obtained from PDB 8VUW. These were used to perform real space refinement in PHENIX 1.19.2 [42]. The structure of ELIC was manually built into the cryo-EM density map using COOT 0.9.6 [43], refined in PHENIX, and this process was repeated iteratively. Propylamine was fit into the density in the agonist binding site based on the orientation predicted from MD simulations [35]. For all C5 models, a single subunit was built, refined and propagated to the other subunits. 3D volume visualization, analysis and image preparation were performed using PyMOL 2.5.2 and ChimeraX 1.6.1.

The fluorescence stopped-flow liposomal flux measurements were performed on the SX20 stopped-flow spectrofluorometer (Applied Photophysics) as previously described [15], but with some modifications to assay ELIC activity in liposomes formed by dialysis—the same liposome reconstitution method that was used for cryo-EM. 2:1:1 POPC:POPE:POPG lipids were dried and reconstituted in 10 mM HEPES pH 7, 100 mM NaNO₃, 1% cholate at 5 mg/ml and sonicated to clarity. For each sample, 2.5 mg of lipid (0.5 ml) was used and mixed with 12.5 μg of WT ELIC or 2.5 μg of ELIC5, incubated for 1 h at 4 °C, and dialyzed for 3-5 days against 14 ml of buffer (50 mM HEPES pH 7, 100 mM NaNO₃) with 6 solution changes at 4°C. Next, the proteoliposome samples were mixed with 1.5 ml of 50 mM HEPES pH 7, 100 mM NaNO₃, and 75 mM 8-Aminonaphthalene-1,3,6,-Trisulfonic Acid (ANTS, ThermoFisher Scientific). The samples underwent a single freeze-thaw to introduce ANTS into the intra-liposomal space. To remove the extra-liposomal ANTS, the samples were diluted to 2.5 ml and run through a PD-10 desalting column (Cytiva) pre-equilibrated with 50 mM HEPES pH 7, 140 mM NaNO₃ (Stopped-flow Buffer A), and eluted with 3 ml of this buffer. Lastly, the samples were diluted 3-fold with Stopped-flow Buffer A.

Two experiments were performed to assess activation and desensitization of WT ELIC, and activation of ELIC5. For WT ELIC, activation and desensitization proceed in the millisecond and second time scales, respectively, so a rapid sequential-mixing experiment was performed with delay times from 10 ms to 60 s. WT ELIC proteoliposomes were mixed 1:1 with Stopped-flow Buffer A containing 2x concentration of propylamine (20 mM). After a variable delay time, the sample was mixed 1:1 with quenching buffer (50 mM HEPES pH 7, 90 mM NaNO₃, 50 mM TlNO₃), and ANTS fluorescence was recorded for 1 s. Multiple repeat measurements were obtained for each delay time and averaged. For ELIC5, activation proceeds in the seconds time scale and the channel is anticipated to not desensitize. To test whether ELIC5 exhibits any desensitization over a long time scale (up to 20 minutes) we modified the assay to add propylamine manually. At predetermined times (1-20 minutes), a single mixing experiment was performed that involved 1:1 mixing with quenching buffer followed by measurement of ANTS fluorescence for 1 s. The data were analyzed using the Pro-Data SX software from Applied Photophysics. The fluorescence quenching traces were fit to a stretched exponential and the rate of Tl⁺ influx was determined at 2 ms [44].