Search

We describe molecular-level functional changes in the α4β2 nicotinic acetylcholine receptor by a leucine residue insertion in the M2 transmembrane domain of the α4 subunit associated with sleep-related hyperkinetic epilepsy. Measurements of agonist-elicited single-channel currents reveal the primary effect is to stabilize the open channel state, while the secondary effect is to promote reopening of the channel. These dual effects prolong the durations of bursts of channel openings equally for the two major stoichiometric forms of the receptor, (α4) 2 (β2) 3 and (α4) 3 (β2) 2 , indicating the functional impact is independent of mutant copy number per receptor. Altering the location of the residue insertion within M2 shows that functionally pivotal structures are confined to a half turn of the M2 α-helix. Residue substitutions within M2 and surrounding α-helices reveal that both intrasubunit and intersubunit interactions mediate the increase in burst duration. These interactions impacting burst duration depend linearly on the size and hydrophobicity of the substituting residue. Together, the results reveal a novel structural region of the α4β2 nicotinic acetylcholine receptor in which interhelical interactions tune the stability of the open channel state., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Epithelial Na + channels (ENaCs) and related channels have large extracellular domains where specific factors interact and induce conformational changes, leading to altered channel activity. However, extracellular structural transitions associated with changes in ENaC activity are not well defined. Using crosslinking and two-electrode voltage clamp in Xenopus oocytes, we identified several pairs of functional intersubunit contacts where mouse ENaC activity was modulated by inducing or breaking a disulfide bond between introduced Cys residues. Specifically, crosslinking E499C in the β-subunit palm domain and N510C in the α-subunit palm domain activated ENaC, whereas crosslinking βE499C with αQ441C in the α-subunit thumb domain inhibited ENaC. We determined that bridging βE499C to αN510C or αQ441C altered the Na + self-inhibition response via distinct mechanisms. Similar to bridging βE499C and αQ441C, we found that crosslinking palm domain αE557C with thumb domain γQ398C strongly inhibited ENaC activity. In conclusion, we propose that certain residues at specific subunit interfaces form microswitches that convey a conformational wave during ENaC gating and its regulation., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

The outermost lipid-exposed α-helix (M4) in each of the homologous α, β, δ, and γ/ε subunits of the muscle nicotinic acetylcholine receptor (nAChR) has previously been proposed to act as a lipid sensor. However, the mechanism by which this sensor would function is not clear. To explore how the M4 α-helix from each subunit in human adult muscle nAChR influences function, and thus explore its putative role in lipid sensing, we functionally characterized alanine mutations at every residue in αM4, βM4, δM4, and εM4, along with both alanine and deletion mutations in the post-M4 region of each subunit. Although no critical interactions involving residues on M4 or in post-M4 were identified, we found that numerous mutations at the M4-M1/M3 interface altered the agonist-induced response. In addition, homologous mutations in M4 in different subunits were found to have different effects on channel function. The functional effects of multiple mutations either along M4 in one subunit or at homologous positions of M4 in different subunits were also found to be additive. Finally, when characterized in both Xenopus oocytes and human embryonic kidney 293T cells, select αM4 mutations displayed cell-specific phenotypes, possibly because of the different membrane lipid environments. Collectively, our data suggest different functional roles for the M4 α-helix in each heteromeric nAChR subunit and predict that lipid sensing involving M4 occurs primarily through the cumulative interactions at the M4-M1/M3 interface, as opposed to the alteration of specific interactions that are critical to channel function., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

The epithelial Na + channel (ENaC)/degenerin family has a similar extracellular architecture, where specific regulatory factors interact and alter channel gating behavior. The extracellular palm domain serves as a key link to the channel pore. In this study, we used cysteine-scanning mutagenesis to assess the functional effects of Cys-modifying reagents on palm domain β10 strand residues in mouse ENaC. Of the 13 ENaC α subunit mutants with Cys substitutions examined, only mutants at sites in the proximal region of β10 exhibited changes in channel activity in response to methanethiosulfonate reagents. Additionally, Cys substitutions at three proximal sites of β and γ subunit β10 strands also rendered mutant channels methanethiosulfonate-responsive. Moreover, multiple Cys mutants were activated by low concentrations of thiophilic Cd 2+ . Using the Na + self-inhibition response to assess ENaC gating behavior, we identified four α, two β, and two γ subunit β10 strand mutations that changed the Na + self-inhibition response. Our results suggest that the proximal regions of β10 strands in all three subunits are accessible to small aqueous compounds and Cd 2+ and have a role in modulating ENaC gating. These results are consistent with a structural model of mouse ENaC that predicts the presence of aqueous tunnels adjacent to the proximal part of β10 and with previously resolved structures of a related family member where palm domain structural transitions were observed with channels in an open or closed state., Competing Interests: Conflict of interests The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Ryanodine receptors (RyRs) are ion channels that mediate the release of Ca 2+ from the sarcoplasmic reticulum/endoplasmic reticulum, mutations of which are implicated in a number of human diseases. The adjacent C-terminal domains (CTDs) of cardiac RyR (RyR2) interact with each other to form a ring-like tetrameric structure with the intersubunit interface undergoing dynamic changes during channel gating. This mobile CTD intersubunit interface harbors many disease-associated mutations. However, the mechanisms of action of these mutations and the role of CTD in channel function are not well understood. Here, we assessed the impact of CTD disease-associated mutations P4902S, P4902L, E4950K, and G4955E on Ca 2+ - and caffeine-mediated activation of RyR2. The G4955E mutation dramatically increased both the Ca 2+ -independent basal activity and Ca 2+ -dependent activation of [ 3 H]ryanodine binding to RyR2. The P4902S and E4950K mutations also increased Ca 2+ activation but had no effect on the basal activity of RyR2. All four disease mutations increased caffeine-mediated activation of RyR2 and reduced the threshold for activation and termination of spontaneous Ca 2+ release. G4955D dramatically increased the basal activity of RyR2, whereas G4955K mutation markedly suppressed channel activity. Similarly, substitution of P4902 with a negatively charged residue (P4902D), but not a positively charged residue (P4902K), also dramatically increased the basal activity of RyR2. These data suggest that electrostatic interactions are involved in stabilizing the CTD intersubunit interface and that the G4955E disease mutation disrupts this interface, and thus the stability of the closed state. Our studies shed new insights into the mechanisms of action of RyR2 CTD disease mutations., Competing Interests: Conflict of interest W. G. is a recipient of the Alberta Innovates-Health Solutions Graduate Studentship Award; J. W. is a recipient of the Libin Cardiovascular Institute and Cumming School of Medicine Postdoctoral Fellowship Award; B. S. is a recipient of the Heart and Stroke Foundation of Canada Junior Fellowship Award and the Alberta Innovates-Health Solutions Fellowship Award; and S. R. W. C. is the Heart and Stroke Foundation Chair in cardiovascular research. All other authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Highly conserved amino acids are generally anticipated to have similar functions across a protein superfamily, including that of the P2X ion channels, which are gated by extracellular ATP. However, whether and how these functions are conserved becomes less clear when neighboring amino acids are not conserved. Here, we investigate one such case, focused on the highly conserved residue from P2X4, E118 (rat P2X4 numbering, rP2X4), a P2X subtype associated with human neuropathic pain. When we compared the crystal structures of P2X4 with those of other P2X subtypes, including P2X3, P2X7, and AmP2X, we observed a slightly altered side-chain orientation of E118. We used protein chimeras, double-mutant cycle analysis, and molecular modeling to reveal that E118 forms specific contacts with amino acids in the "beak" region, which facilitates ATP binding to rP2X4. These contacts are not present in other subtypes because of sequence variance in the beak region, resulting in decoupling of this conserved residue from ATP recognition and/or channel gating of P2X receptors. Our study provides an example of a conserved residue with a specific role in functional proteins enabled by adjacent nonconserved residues. The unique role established by the E118-beak region contact provides a blueprint for the development of subtype-specific inhibitors of P2X4., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

The activity of the muscle-type Torpedo nicotinic acetylcholine receptor (nAChR) is highly sensitive to lipids, but the underlying mechanisms remain poorly understood. The nAChR transmembrane α-helix, M4, is positioned at the perimeter of each subunit in direct contact with lipids and likely plays a central role in lipid sensing. To gain insight into the mechanisms underlying nAChR lipid sensing, we used homology modeling, coevolutionary analyses, site-directed mutagenesis, and electrophysiology to examine the role of the α-subunit M4 (αM4) in the function of the adult muscle nAChR. Ala substitutions for most αM4 residues, including those in clusters of polar residues at both the N and C termini, and deletion of up to 11 C-terminal residues had little impact on the agonist-induced response. Even Ala substitutions for coevolved pairs of residues at the interface between αM4 and the adjacent helices, αM1 and αM3, had little effect, although some impaired nAChR expression. On the other hand, Ala substitutions for Thr422 and Arg429 caused relatively large losses of function, suggesting functional roles for these specific residues. Ala substitutions for aromatic residues at the αM4-αM1/αM3 interface generally led to gains of function, as previously reported for the prokaryotic homolog, the Erwinia chrysanthemi ligand-gated ion channel (ELIC). The functional effects of individual Ala substitutions in αM4 were found to be additive, although not in a completely independent manner. Our results provide insight into the structural features of αM4 that are important. They also suggest how lipid-dependent changes in αM4 structure ultimately modify nAChR function., Competing Interests: Conflict of interest—The authors declare that they have no conflicts of interest with the contents of this article., (© 2020 Thompson et al.)

The neuropilin and tolloid-like (Neto) proteins Neto1 and Neto2 are auxiliary subunits of kainate-type glutamate receptors (KARs) that regulate KAR trafficking and gating. However, how Netos bind and regulate the biophysical functions of KARs remains unclear. Here, we found that the N-terminal domain (NTD) of glutamate receptor ionotropic kainate 2 (GluK2) binds the first complement C1r/C1s-Uegf-BMP (CUB) domain of Neto proteins ( i.e. NTD-CUB1 interaction) and that the core of GluK2 (GluK2ΔNTD) binds Netos through domains other than CUB1s (core-Neto interaction). Using electrophysiological analysis in HEK293T cells, we examined the effects of these interactions on GluK2 gating, including deactivation, desensitization, and recovery from desensitization. We found that NTD deletion does not affect GluK2 fast gating kinetics, the desensitization, and the deactivation. We also observed that Neto1 and Neto2 differentially regulate GluK2 fast gating kinetics, which largely rely on the NTD-CUB1 interactions. NTD removal facilitated GluK2 recovery from desensitization, indicating that the NTD stabilizes the GluK2 desensitization state. Co-expression with Neto1 or Neto2 also accelerated GluK2 recovery from desensitization, which fully relied on the NTD-CUB1 interactions. Moreover, we demonstrate that the NTD-CUB1 interaction involves electric attraction between positively charged residues in the GluK2_NTD and negatively charged ones in the CUB1 domains. Neutralization of these charges eliminated the regulatory effects of the NTD-CUB1 interaction on GluK2 gating. We conclude that KARs bind Netos through at least two sites and that the NTD-CUB1 interaction critically regulates Neto-mediated GluK2 gating., (© 2019 Li et al.)

The epithelial Na + channel (ENaC) possesses a large extracellular domain formed by a β-strand core enclosed by three peripheral α-helical subdomains, which have been dubbed thumb, finger, and knuckle. Here we asked whether the ENaC thumb domains play specific roles in channel function. To this end, we examined the characteristics of channels lacking a thumb domain in an individual ENaC subunit (α, β, or γ). Removing the γ subunit thumb domain had no effect on Na + currents when expressed in Xenopus oocytes, but moderately reduced channel surface expression. In contrast, ENaCs lacking the α or β subunit thumb domain exhibited significantly reduced Na + currents along with a large reduction in channel surface expression. Moreover, channels lacking an α or γ thumb domain exhibited a diminished Na + self-inhibition response, whereas this response was retained in channels lacking a β thumb domain. In turn, deletion of the α thumb domain had no effect on the degradation rate of the immature α subunit as assessed by cycloheximide chase analysis. However, accelerated degradation of the immature β subunit and mature γ subunit was observed when the β or γ thumb domain was deleted, respectively. Our results suggest that the thumb domains in each ENaC subunit are required for optimal surface expression in oocytes and that the α and γ thumb domains both have important roles in the channel's inhibitory response to external Na + Our findings support the notion that the extracellular helical domains serve as functional modules that regulate ENaC biogenesis and activity., (© 2018 Sheng et al.)

Store-operated Ca 2+ entry signals are mediated by plasma membrane Orai channels activated through intermembrane coupling with Ca 2+ -sensing STIM proteins in the endoplasmic reticulum (ER). The nature of this elaborate Orai-gating mechanism has remained enigmatic. Based on the Drosophila Orai structure, mammalian Orai1 channels are hexamers comprising three dimeric subunit pairs. We utilized concatenated Orai1 dimers to probe the function of key domains within the channel pore and gating regions. The Orai1-E106Q selectivity-filter mutant, widely considered a dominant pore blocker, was surprisingly nondominant within concatenated heterodimers with Orai1-WT. The Orai1-E106Q/WT heterodimer formed STIM1-activated nonselective cation channels with significantly enlarged apparent pore diameter. Other Glu-106 substitutions entirely blocked the function of heterodimers with Orai1-WT. The hydrophobic pore-lining mutation V102C, which constitutively opens channels, was suppressed by Orai1-WT in the heterodimer. In contrast, the naturally occurring R91W pore-lining mutation associated with human immunodeficiency was completely dominant-negative over Orai-WT in heterodimers. Heterodimers containing the inhibitory K85E mutation extending outward from the pore helix gave an interesting partial effect on both channel activation and STIM1 binding, indicating an important allosteric link between the cytosolic Orai1 domains. The Orai1 C-terminal STIM1-binding domain mutation L273D powerfully blocked STIM1-induced channel activation. The Orai1-L273D/WT heterodimer had drastically impaired STIM1-induced channel gating but, unexpectedly, retained full STIM1 binding. This reveals the critical role of Leu-273 in transducing the STIM1-binding signal into the allosteric conformational change that initiates channel gating. Overall, our results provide important new insights into the role of key functional domains that mediate STIM1-induced gating of the Orai1 channel., (© 2018 Cai et al.)

FMRFamide (Phe-Met-Arg-Phe-NH2)-activated sodium channel (FaNaC) is an amiloride-sensitive sodium channel activated by endogenous tetrapeptide in invertebrates, and belongs to the epithelial sodium channel/degenerin (ENaC/DEG) superfamily. The ENaC/DEG superfamily differs markedly in its means of activation, such as spontaneously opening or gating by mechanical stimuli or tissue acidosis. Recently, it has been observed that a number of ENaC/DEG channels can be activated by small molecules or peptides, indicating that the ligand-gating may be an important feature of this superfamily. The peptide ligand control of the channel gating might be an ancient ligand-gating feature in this superfamily. Therefore, studying the peptide recognition of FaNaC channels would advance our understanding of the ligand-gating properties of this superfamily of ion channels. Here we demonstrate that Tyr-131, Asn-134, Asp-154, and Ile-160, located in the putative upper finger domain ofHelix aspersaFaNaC (HaFaNaC) channels, are key residues for peptide recognition of this ion channel. Two HaFaNaC specific-insertion motifs among the ENaC/DEG superfamily, residing at the putative α4-α5 linker of the upper thumb domain and the α6-α7 linker of the upper knuckle domain, are also essential for the peptide recognition of HaFaNaC channels. Chemical modifications and double mutant cycle analysis further indicated that those two specific inserts and key residues in the upper finger domain together participate in peptide recognition of HaFaNaC channels. This ligand recognition site is distinct from that of acid-sensing ion channels (ASICs) by a longer distance between the recognition site and the channel gate, carrying useful information about the ligand gating and the evolution of the trimeric ENaC/DEG superfamily of ion channels., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

The voltage-gated H(+) channel (Hv) is a voltage sensor domain-like protein consisting of four transmembrane segments (S1-S4). The native Hv structure is a homodimer, with the two channel subunits functioning cooperatively. Here we show that the two voltage sensor S4 helices within the dimer directly cooperate via a π-stacking interaction between Trp residues at the middle of each segment. Scanning mutagenesis showed that Trp situated around the original position provides the slow gating kinetics characteristic of the dimer's cooperativity. Analyses of the Trp mutation on the dimeric and monomeric channel backgrounds and analyses with tandem channel constructs suggested that the two Trp residues within the dimer are functionally coupled during Hv deactivation but are less so during activation. Molecular dynamics simulation also showed direct π-stacking of the two Trp residues. These results provide new insight into the cooperative function of voltage-gated channels, where adjacent voltage sensor helices make direct physical contact and work as a single unit according to the gating process., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

KirBac channels are prokaryotic homologs of mammalian inwardly rectifying potassium (Kir) channels, and recent structures of KirBac3.1 have provided important insights into the structural basis of gating in Kir channels. In this study, we demonstrate that KirBac3.1 channel activity is strongly pH-dependent, and we used x-ray crystallography to determine the structural changes that arise from an activatory mutation (S205L) located in the cytoplasmic domain (CTD). This mutation stabilizes a novel energetically favorable open conformation in which changes at the intersubunit interface in the CTD also alter the electrostatic potential of the inner cytoplasmic cavity. These results provide a structural explanation for the activatory effect of this mutation and provide a greater insight into the role of the CTD in Kir channel gating.

TRPC4 and TRPC5 proteins share 65% amino acid sequence identity and form Ca(2+)-permeable nonselective cation channels. They are activated by stimulation of receptors coupled to the phosphoinositide signaling cascade. Replacing a conserved glycine residue within the cytosolic S4-S5 linker of both proteins by a serine residue forces the channels into an open conformation. Expression of the TRPC4G503S and TRPC5G504S mutants causes cell death, which could be prevented by buffering the Ca(2+) of the culture medium. Current-voltage relationships of the TRPC4G503S and TRPC5G504S mutant ion channels resemble that of fully activated TRPC4 and TRPC5 wild-type channels, respectively. Modeling the structure of the transmembrane domains and the pore region (S4-S6) of TRPC4 predicts a conserved serine residue within the C-terminal sequence of the predicted S6 helix as a potential interaction site. Introduction of a second mutation (S623A) into TRPC4G503S suppressed the constitutive activation and partially rescued its function. These results indicate that the S4-S5 linker is a critical constituent of TRPC4/C5 channel gating and that disturbance of its sequence allows channel opening independent of any sensor domain.

In co-translational translocation, the ribosome funnel and the channel of the protein translocation complex SecYEG are aligned. For the nascent chain to enter the channel immediately after synthesis, a yet unidentified signal triggers displacement of the SecYEG sealing plug from the pore. Here, we show that ribosome binding to the resting SecYEG channel triggers this conformational transition. The purified and reconstituted SecYEG channel opens to form a large ion-conducting channel, which has the conductivity of the plug deletion mutant. The number of ion-conducting channels inserted into the planar bilayer per fusion event roughly equals the number of SecYEG channels counted by fluorescence correlation spectroscopy in a single proteoliposome. Thus, the open probability of the channel must be close to unity. To prevent the otherwise lethal proton leak, a closed post-translational conformation of the SecYEG complex bound to a ribosome must exist.