M2 Helix Research Articles

Clinical phenotype and functional influence of GRIN2A variants in epilepsy-aphasia syndrome.

N-methyl-D-aspartate receptors are glutamate-gated ion channels that play a crucial role in brain function. Numerous inherited or de novo variants in the GRIN2A gene, encoding the GluN2A subunit of the receptor, have been identified in patients with epilepsy. In addition, it is worth noting that GRIN2A variants exhibit a strong correlation with epilepsy-aphasia syndromes, a group of age-dependent epileptic, cognitive, and language disorders with a characteristic electroencephalographic pattern. Whole exome sequencing was conducted in enrolled patients with epilepsy-aphasia syndromes, and GRIN2A variants were screened. The conservation of substituted residues, conformational changes of mutant subunits, and in silico predictions of pathogenicity were thoroughly assessed in our study. Functional alterations of the variants were examined using whole-cell voltage-clamp current recordings while the relative surface expression levels of subunit proteins were assessed via immunofluorescence assays. A summary of previously published GRIN2A missense variants was conducted to investigate the genotypic-phenotypic-functional correlations. Two missense GRIN2A variants (c. 2482A >G/p. M828V, c.2627 T >C/p. I876T) were identified, which are located in the transmembrane helix M4 and C-terminus domain of the GluN2A subunit, respectively. Both variants exhibited reduced current density of NMDARs and surface/total expression levels of GluN2A subunits, while M828V showed a decreased extent of desensitization as well. A further summary of the previously reported GRIN2A variants demonstrated that more variable phenotypes were observed for variants situated in the C-terminus domain or those with loss-of-function effects. Our study expands the phenotypic and functional range of GRIN2A-related disorders. In order to optimally establish the domain-function-phenotype correlations in GRIN2A variants, it is imperative to gather a more extensive set of clinical and functional data. This study has identified two genetic variants of the GRIN2A gene in patients with epilepsy-aphasia syndrome. We assess the variants' harmfulness through a variety of functional experiments, including evaluating the expression level of the mutated protein and the resulting changes in electrophysiological activities. Also, we reviewed previously published papers about GRIN2A variants in epilepsy to learn more about the correlations between their locations, functional changes, and clinical manifestations.

Grin1 Y 647 S/+ Mice: A Preclinical Model of GRIN1 -Related Neurodevelopmental Disorder.

GRIN1 -related neurodevelopmental disorder ( GRIN1 -NDD) is characterized by clinically significant variation in the GRIN1 gene, which encodes the obligatory GluN1 subunit of N-methyl-D-aspartate receptors (NMDARs). The identified p.Tyr647Ser (Y647S) variant - carried by a 33-year-old female with seizures and intellectual disability - is located in the M3 helix in the GluN1 transmembrane domain. This study builds upon initial in vitro investigations of the functional impacts of the GRIN1 Y647S variant and examines its in vivo consequences in a mouse model. To investigate in vitro functional impacts of NMDARs containing GluN1-Y647S variant subunits, GluN1-Y647S was co-expressed with wildtype GluN2A or GluN2B subunits in Xenopus laevis oocytes and HEK cells. Grin1 Y647S/+ mice were created by CRISPR-Cas9 endonuclease-mediated transgenesis and the molecular, electrophysiological, and behavioural consequences of the variant were examined. In vitro , NMDARs containing GluN1-Y647S show altered sensitivity to endogenous agonists and negative allosteric modulators, and reduced cell surface trafficking. Grin1 Y647S/+ mice displayed a reduction in whole brain GluN1 levels and deficiency in NMDAR-mediated synaptic transmission in the hippocampus. Behaviourally, Grin1 Y647S/+ mice exhibited spontaneous seizures, altered vocalizations, muscle strength, sociability, and problem-solving. The Y647S variant confers a complex in vivo phenotype, which reflects largely diminished properties of NMDAR function. As a result, Grin1 Y647S/+ mice display atypical behaviour in domains relevant to the clinical characteristics of GRIN1 -NDD and the individual carrying the variant. Ultimately, the characterization of Grin1 Y647S/+ mice accomplished in the present work expands our understanding of the mechanisms underlying GRIN1 -NDD and provides a foundation for the development of novel therapeutics.

Kainate receptor channel opening and gating mechanism

Kainate receptors, a subclass of ionotropic glutamate receptors, are tetrameric ligand-gated ion channels that mediate excitatory neurotransmission1–4. Kainate receptors modulate neuronal circuits and synaptic plasticity during the development and function of the central nervous system and are implicated in various neurological and psychiatric diseases, including epilepsy, depression, schizophrenia, anxiety and autism5–11. Although structures of kainate receptor domains and subunit assemblies are available12–18, the mechanism of kainate receptor gating remains poorly understood. Here we present cryo-electron microscopy structures of the kainate receptor GluK2 in the presence of the agonist glutamate and the positive allosteric modulators lectin concanavalin A and BPAM344. Concanavalin A and BPAM344 inhibit kainate receptor desensitization and prolong activation by acting as a spacer between the amino-terminal and ligand-binding domains and a stabilizer of the ligand-binding domain dimer interface, respectively. Channel opening involves the kinking of all four pore-forming M3 helices. Our structures reveal the molecular basis of kainate receptor gating, which could guide the development of drugs for treatment of neurological disorders.

De novo GRIN variants in M3 helix associated with neurological disorders control channel gating of NMDA receptor

N-methyl-d-aspartate receptors (NMDARs) are members of the glutamate receptor family and participate in excitatory postsynaptic transmission throughout the central nervous system. Genetic variants in GRIN genes encoding NMDAR subunits are associated with a spectrum of neurological disorders. The M3 transmembrane helices of the NMDAR couple directly to the agonist-binding domains and form a helical bundle crossing in the closed receptors that occludes the pore. The M3 functions as a transduction element whose conformational change couples ligand binding to opening of an ion conducting pore. In this study, we report the functional consequences of 48 de novo missense variants in GRIN1, GRIN2A, and GRIN2B that alter residues in the M3 transmembrane helix. These de novo variants were identified in children with neurological and neuropsychiatric disorders including epilepsy, developmental delay, intellectual disability, hypotonia and attention deficit hyperactivity disorder. All 48 variants in M3 for which comprehensive testing was completed produce a gain-of-function (28/48) compared to loss-of-function (9/48); 11 variants had an indeterminant phenotype. This supports the idea that a key structural feature of the M3 gate exists to stabilize the closed state so that agonist binding can drive channel opening. Given that most M3 variants enhance channel gating, we assessed the potency of FDA-approved NMDAR channel blockers on these variant receptors. These data provide new insight into the structure–function relationship of the NMDAR gate, and suggest that variants within the M3 transmembrane helix produce a gain-of-function.

Mechanism of hydrophobic gating in the acetylcholine receptor channel pore.

Neuromuscular acetylcholine receptors (AChRs) are hetero-pentameric, ligand-gated ion channels. The binding of the neurotransmitter acetylcholine (ACh) to two target sites promotes a global conformational change of the receptor that opens the channel and allows ion conduction through the channel pore. Here, by measuring free-energy changes from single-channel current recordings and using molecular dynamics simulations, we elucidate how a constricted hydrophobic region acts as a "gate" to regulate the channel opening in the pore of AChRs. Mutations of gate residues, including those implicated in congenital myasthenia syndrome, lower the permeation barrier of the channel substantially and increase the unliganded gating equilibrium constant (constitutive channel openings). Correlations between hydrophobicity and the observed free-energy changes, supported by calculations of water densities in the wild-type versus mutant channel pores, provide evidence for hydrophobic wetting-dewetting transition at the gate. The analysis of a coupled interaction network provides insight into the molecular mechanism of closed- versus open-state conformational changes at the gate. Studies of the transition state by "phi"(φ)-value analysis indicate that agonist binding serves to stabilize both the transition and the open state. Intersubunit interaction energy measurements and molecular dynamics simulations suggest that channel opening involves tilting of the pore-lining M2 helices, asymmetric outward rotation of amino acid side chains, and wetting transition of the gate region that lowers the barrier to ion permeation and stabilizes the channel open conformation. Our work provides new insight into the hydrophobic gate opening and shows why the gate mutations result in constitutive AChR channel activity.

Subunit gating resulting from individual protonation events in Kir2 channels

Inwardly rectifying potassium (Kir) channels open at the ‘helix bundle crossing’ (HBC), formed by the M2 helices at the cytoplasmic end of the transmembrane pore. Introduced negative charges at the HBC (G178D) in Kir2.2 channels forces opening, allowing pore wetting and free movement of permeant ions between the cytoplasm and the inner cavity. Single-channel recordings reveal striking, pH-dependent, subconductance behaviors in G178D (or G178E and equivalent Kir2.1[G177E]) mutant channels, with well-resolved non-cooperative subconductance levels. Decreasing cytoplasmic pH shifts the probability towards lower conductance levels. Molecular dynamics simulations show how protonation of Kir2.2[G178D], or the D173 pore-lining residues, changes solvation, K+ ion occupancy, and K+ conductance. Ion channel gating and conductance are classically understood as separate processes. The present data reveal how individual protonation events change the electrostatic microenvironment of the pore, resulting in step-wise alterations of ion pooling, and hence conductance, that appear as ‘gated’ substates.

Two gates mediate NMDA receptor activity and are under subunit-specific regulation

Kinetics of NMDA receptor (NMDAR) ion channel opening and closing contribute to their unique role in synaptic signaling. Agonist binding generates free energy to open a canonical gate at the M3 helix bundle crossing. Single channel activity is characterized by clusters, or periods of rapid opening and closing, that are separated by long silent periods. A conserved glycine in the outer most transmembrane helices, the M4 helices, regulates NMDAR function. Here we find that the GluN1 glycine mainly regulates single channel events within a cluster, whereas the GluN2 glycine mainly regulates entry and exit from clusters. Molecular dynamics simulations suggest that, whereas the GluN2 M4 (along with GluN2 pre-M1) regulates the gate at the M3 helix bundle crossing, the GluN1 glycine regulates a ‘gate’ at the M2 loop. Subsequent functional experiments support this interpretation. Thus, the distinct kinetics of NMDARs are mediated by two gates that are under subunit-specific regulation.

Molecular Dynamics Refinement of Open State Serotonin 5-HT3A Receptor Structures.

Pentameric ligand-gated ion channels play an important role in mediating fast neurotransmissions. As a member of this receptor family, cation-selective 5-HT3 receptors are a clinical target for treating nausea and vomiting associated with chemotherapy and radiation therapy (Thompson and Lummis, 2006). Multiple cryo-electron microscopy (cryo-EM) structures of 5-HT3 receptors have been determined in distinct functional states (e.g., open, closed, etc.) (Basak et al., 2018; Basak et al., 2018; Polovinkin et al., 2018; Zhang et al., 2015). However, recent work has shown that the transmembrane pores of the open 5-HT3 receptor structures rapidly collapse and become artificially asymmetric in molecular dynamics (MD) simulations. To avoid this hydrophobic collapse, Dämgen and Biggin developed an equilibration protocol that led to a stable open state structure of the glycine receptor in MD simulations (Dämgen and Biggin, 2020). However, the protocol failed to yield open-like structures of the 5-HT3 receptor in our simulations. Here, we present a refined equilibration protocol that involves the rearrangement of the transmembrane helices to achieve stable open state structures of the 5-HT3 receptor that allow both water and ion permeation through the channel. Notably, channel gating is mediated through collective movement of the transmembrane helices, involving not only pore lining M2 helices but also their cross-talk with the adjacent M1 and M3 helices. Thus, the successful application of our refined equilibration protocol underscores the importance of the conformational coupling between the transmembrane helices in stabilizing open-like structures of the 5-HT3 receptor.

Sodium pump regulation in health and disease.

The sodium-potassium ATPase (NKA) is the ion motive ATP-dependent transporter that establishes Na+ and K+ gradients across the cell membrane, facilitating many physiological processes. In the heart, NKA activity is regulated by phospholemman (PLM), which PLM inhibits NKA by reducing its apparent affinity for Na+. In this study, we used time-correlated single-photon counting, FRET-microscopy, molecular dynamics simulations, and electrophysiology to investigate the stoichiometry of the native NKA-PLM regulatory complex. Our data suggest a stoichiometry of the NKA-PLM complex composed of two α subunits, two β subunits, and decorated with two PLM regulators. Protein-protein docking and molecular dynamics simulations generated a structural model of the regulatory complex. This model was characterized by contacts between cytoplasmic domains of α subunits, between β subunit extracellular domains, and between the highly-conserved α subunits’ M3 helix and the N-termini of opposing β subunit. The helix M3 shows significant movement during the pump's reaction cycle and mutations in M3 are associated with several diseases. Here, we studied the G301R mutant that is unable to correctly localize in the plasma membrane when expressed alone, but a milder G301A mutant localizes correctly. The G301R localization was improved in the presence of a WT pump suggesting a dominant negative effect of this mutation. Our simulations of the dimeric complexes revealed decreased affinity of the Na+ in position II, which is unbound during the simulated time window. Surprisingly, Na+ binding was not affected in the simulated WT or G301A/R monomers. Two-electrode voltage clamp experiments revealed decreased affinity of G301A for external Na+ and ion leak from G301R mutants supporting our simulation data. This combination of ex vivo and in silico experiments provides an insight into the quaternary structure of the NKA-PLM regulatory complex and helps to explain the mechanism of G301A/R mutation pathology.

Two gates mediate NMDAR activity and are under subunit-specific regulation.

NMDA receptors (NMDAR) are ionotropic glutamate receptors that mediate fast synaptic transmission and synaptic plasticity. Dysfunction of NMDARs has been found in numerous neurological diseases including Alzheimer's disease, Intellectual disability, epilepsy, autism, and schizophrenia. The NMDARs broad role in neuronal function is reflected in the complexity of its opening and closing kinetics, which can be broadly divided into periods of high opening activity, or clusters, interspersed by periods of no activity, or inter clusters. The initial step in ion channel opening is binding of agonists to the ligand-binding domain (LBD), which generates free energy that propagates to the ion channel to open ‘gate(s)’. NMDARs are heterotetramers composed of the obligate GluN1 and typically some combination of GluN2 subunits. The ion channel pore consists of two transmembrane segments (M1 and M3), a reentrant pore loop (M2) and a peripheral M4. A conserved glycine in the outer most transmembrane helix, the M4 helix, plays important roles in regulating NMDAR function. Here we find that these glycines play different roles in regulating channel opening. The GluN1 glycine mainly regulates single channel events within a cluster or bursts of activity, whereas GluN2A glycine mainly regulates opening into and out of clusters. Molecular dynamic simulations suggest that the GluN1 M4 regulates a ‘gate’ at the M2 loop, whereas the GluN2 M4 (along with GluN2 pre-M1) regulates a gate at the M3 helix bundle crossing. Subsequent functional experiments support this interpretation, leading to the hypothesis that the distinct kinetics of NMDARs are mediated by two gates that are under subunit-specific regulation. These results indicate that the GluN2A subunit regulates early steps in the gating process whereas the GluN1 regulate later steps including transitions form short to long-lived open states.

Uncovering individual protonation events in Kir2 channels.

Inwardly rectifying potassium (Kir) channels play a critical role in stabilizing the membrane potential, thus controlling numerous physiological phenomena in various tissues. Channel conductance can be activated by cytoplasmic modulators that open the channel at the ‘helix bundle crossing’ (HBC), formed by the coming together of the M2 helix from each of the four subunits, at the cytoplasmic end of the transmembrane pore.

The role of zinc binding sites in the cation diffusion facilitator YiiP.

YiiP is a zinc/proton antiporter that couples zinc export to the proton motive force. YiiP binds zinc at three sites: site A in the transmembrane domain, site B in a cytoplasmic loop between transmembrane helices M2 and M3, and site C in the cytoplasmic, C-terminal domain (CTD). Previous Cryo-EM analysis revealed that global removal of zinc by EDTA produced a novel inward occluded state that was postulated to be an intermediate between the inward and outward facing states. In this study we introduced mutations to study binding affinities and conformational effects of each individual site. Cryo-EM analysis showed that disruption of site A produces little change, but that disruption of site B induces the inward occluded state observed previously. Double mutation at site C produced nonnative oligomers, indicating that site C is important for assembly of the native homodimer. Molecular dynamic simulations with zinc removed from site B showed increased mobility of the CTD and disordering of the M2M3 loop in agreement with the structural studies. Binding affinity was quantified by microscale thermophoresis using either citrate or NTA to buffer the concentration of free zinc. The results demonstrate nanomolar affinity at sites A and C and micromolar affinity at site B. Site A is highly sensitive to pH changes with Kd increasing from 1 nM to 302 μM as pH decreases from 7.4 to 5.6. Based on these observations, we propose a transport mechanism whereby zinc is initially recruited at site B and that transfer to site A induces conformational changes leading to transport. The pH dependence of site A is key to energy coupling by ensuring high affinity binding from the cytoplasm and then promoting release to the more acidic periplasmic milieau.

Using fluorescence to measure real-time binding of serotonin to its receptors.

Neurotransmitter binding assays that can be performed on or near physiological time scales would greatly enhance our understanding of nervous signaling. We set out to create one such assay to measure the binding of serotonin (5-hydroxytryptamine, 5-HT) to intact, fluorescently-tagged receptors. Tryptophan quenches the small organic fluorophores Prodan and bimane via photo-induced electron transfer. As serotonin shares a common indole structure with tryptophan, we reasoned that it may also quench the Prodan-based fluorescent amino acid L-3-(6-acetylnaphthalen-2-ylamino)-2-aminopropionic acid (ANAP). In solution, serotonin quenches ANAP in the mM range. We used amber stop-codon suppression to express ionotropic 5-HT3A serotonin receptors tagged with ANAP in the serotonin binding site in HEK293T cells. To ensure that our protein constructs were not prematurely truncated at the amber stop codon, we included a downstream mOrange protein tag in the cytoplasmic loop between the M3 and M4 transmembrane helices. We used a microscope-coupled spectrograph to acquire fluorescence emission spectra from ANAP-tagged channels and found that the addition of 5-HT reduced ANAP fluorescence in a dose-dependent fashion at concentrations in the low µM range. Based on our findings, we believe this strategy offers great promise for the detection of 5-HT binding to functional receptors in intact cells. Such measurements can be paired with voltage clamp to study the binding-activity relationship of 5-HT3A and may also offer insights into the function of metabotropic serotonin receptors.

Membrane thickness affects ELIC activity & M4 helix structure.

Pentameric ligand gated ion channels (pLGICs) are a superfamily of ion channels involved in fast neural transmission. Some examples include the GABA(A) receptor and nicotinic acetylcholine receptor (nAchR). In the nAchR, it has been shown that thick membranes promote coupling of agonist binding to channel activation. However, the effect of membrane thickness on the ion transport activity of any pLGIC is not known. To investigate this, we reconstituted the model pLGIC, Erwinia ligand gated ion channel (ELIC), in liposomes consisting of di-monounsaturated phosphatidylcholine (PC) of varying acyl chain length (16-22), and assayed ion flux using a sequential mixing fluorescence quenching assay. ELIC exhibited maximal agonist responses in di-20:1PC liposomes with a dramatic decrease in thinner or thicker membranes. To verify that differences in agonist responses are not a function of changes in channel orientation in the liposomes, we further assayed an ELIC construct with a quenchable fluorescent label in the N-terminal ECD. Minimal differences in the amount of outwardly facing channels were observed in different membrane conditions confirming that changes in agonist responses are primarily a function of channel activity. To investigate the structural basis of ELIC sensitivity to membrane thickness, we obtained apo and agonist-bound structures of ELIC in a circularized nanodisc with di16:1PC, di20:1PC or di22:1PC using single particle cryo-EM. In the agonist-bound structures, the membrane facing helix, M4, is poorly resolved in thin (di16:1PC) and thick (di22:1PC) membranes but clearly resolved in di20:1PC, indicating a correlation between the structure and dynamics of M4 and the optimal membrane thickness that supports channel function. These findings indicate that ELIC function is promoted by thicker membranes and suggests that sensing of membrane thickness is mediated by M4, as has been proposed for the nAchR.

Allosteric modulation of α1β3γ2 GABAA receptors by farnesol through the neurosteroid sites

In mammalian cells, all-trans farnesol, a 15-carbon isoprenol, is a product of the mevalonate pathway. It is the natural substrate of alcohol dehydrogenase and a substrate for CYP2E1, two enzymes implicated in ethanol metabolism. Studies have shown that farnesol is present in the human brain and inhibits voltage-gated Ca2+ channels at much lower concentrations than ethanol. Here we show that farnesol modulates the activity of γ-aminobutyric acid type A receptors (GABAARs), some of which also mediate the sedative activity of ethanol. Electrophysiology experiments performed in HEK cells expressing human α1β3γ2 or α6β3γ2 GABAARs revealed that farnesol increased chloride currents through positive allosteric modulation of these receptors and showed dependence on both the alcoholic functional group of farnesol and the length of the alkyl chain for activity. In silico studies using long-timescale unbiased all-atom molecular dynamics (MD) simulations of the human α1β3γ2 GABAA receptors revealed that farnesol modulates the channel by directly binding to the transmembrane neurosteroid-binding site, after partitioning into the surrounding membrane and reaching the receptor by lateral diffusion. Channel activation by farnesol was further characterized by several structural and dynamic variables, such as global twisting of the receptor’s extracellular domain, tilting of the transmembrane M2 helices, radius, cross-sectional area, hydration status, and electrostatic potential of the channel pore. Our results expand the pharmacological activities of farnesol to yet another class of ion channels implicated in neurotransmission, thus providing a novel path for understanding and treatment of diseases involving GABAA receptor dysfunction.

Structure of human phagocyte NADPH oxidase in the resting state.

Phagocyte oxidase plays an essential role in the first line of host defense against pathogens. It oxidizes intracellular NADPH to reduce extracellular oxygen to produce superoxide anions that participate in pathogen killing. The resting phagocyte oxidase is a heterodimeric complex formed by two transmembrane proteins NOX2 and p22. Despite the physiological importance of this complex, its structure remains elusive. Here, we reported the cryo-EM structure of the functional human NOX2-p22 complex in nanodisc in the resting state. NOX2 shows a canonical 6-TM architecture of NOX and p22 has four transmembrane helices. M3, M4, and M5 of NOX2, and M1 and M4 helices of p22 are involved in the heterodimer formation. Dehydrogenase (DH) domain of NOX2 in the resting state is not optimally docked onto the transmembrane domain, leading to inefficient electron transfer and NADPH binding. Structural analysis suggests that the cytosolic factors might activate the NOX2-p22 complex by stabilizing the DH in a productive docked conformation.

Full opening of helix bundle crossing does not lead to NaK channel activation.

A critical part of ion channel function is the ability to open and close in response to stimuli and thus conduct ions in a regulated fashion. While x-ray diffraction studies of ion channels suggested a general steric gating mechanism located at the helix bundle crossing (HBC), recent functional studies on several channels indicate that the helix bundle crossing is wide-open even in functionally nonconductive channels. Two NaK channel variants were crystallized in very different open and closed conformations, which served as important models of the HBC gating hypothesis. However, neither of these NaK variants is conductive in liposomes unless phenylalanine 92 is mutated to alanine (F92A). Here, we use NMR to probe distances at near-atomic resolution of the two NaK variants in lipid bicelles. We demonstrate that in contrast to the crystal structures, both NaK variants are in a fully open conformation, akin to Ca2+-bound MthK channel structure where the HBC is widely open. While we were not able to determine what a conductive NaK structure is like, our further inquiry into the gating mechanism suggests that the selectivity filter and pore helix are coupled to the M2 helix below and undergo changes in the structure when F92 is mutated. Overall, our data show that NaK exhibits coupling between the selectivity filter and HBC, similar to K+ channels, and has a more complex gating mechanism than previously thought, where the full opening of HBC does not lead to channel activation.

ATP hydrolytic activity of purified Spf1p correlate with micellar lipid fluidity and is dependent on conserved residues in transmembrane helix M1.

P5A ATPases are expressed in the endoplasmic reticulum (ER) of all eukaryotic cells, and their disruption results in pleiotropic phenotypes related to severe ER stress. They were recently proposed to function in peptide translocation although their specificity have yet to be confirmed in reconstituted assays using the purified enzyme. A general theme for P-type ATPases is that binding and transport of substrates is coupled to hydrolysis of ATP in a conserved allosteric mechanism, however several independent reports have shown purified Spf1p to display intrinsic spontaneous ATP hydrolytic activity after purification. It has never been determined to what extend this spontaneous activity is caused by uncoupling of the enzyme. In this work we have purified a functional tagged version of the Saccharomyces cerevisiae P5A ATPase Spf1p and have observed that the intrinsic ATP hydrolytic activity of the purified and re-lipidated protein can be stimulated by specific detergents (C12E8, C12E10 and Tween20) in mixed lipid/detergent micelles in the absence of any apparent substrate. We further show that this increase in activity correlate with the reaction temperature and the anisotropic state of the mixed lipid/detergent micelles and further that this correlation relies on three highly conserved phenylalanine residues in M1. This suggests that at least part of the intrinsic ATP hydrolytic activity is allosterically coupled to movements in the TM domain in the purified preparations. It is suggested that free movement of the M1 helix represent an energetic constraint on catalysis and that this constraint likely is lost in the purified preparations resulting in protein with intrinsic spontaneous ATP hydrolytic activity. Removal of the N-terminal part of the protein apparently removes this activity.

Mutations of the nACh Receptor M4 Helix Reveal Different Phenotypes in Different Expression Systems: Could Lipids be Responsible?

The role of the outermost helix (M4) in the pentameric ligand-gated ion channel (pLGIC) family is currently not fully understood. It is known that M4 is important for receptor assembly, possibly via interactions with neighboring M1 and M3 helices. M4 can also transmit information on the lipid content of the membrane to the gating mechanism, and it may form a link to the extracellular domain via the Cys-loop. Our previous study examining the α4β2 nACh receptor M4 helix using HEK cells indicated M4 here is more sensitive to change than those of other pLGIC. Many of these other studies, however, were performed in Xenopus oocytes. Here we examine the nine previously identified nonfunctional α4β2 nACh receptor M4 mutant receptors using this system. The data reveal that seven of these mutant receptors do function when expressed in oocytes, with only 2, the conserved Asp at the intracellular end of M4 and a Phe in the center, having a similar phenotype (nonfunctional) in both HEK cells and oocytes. The oocyte data are more consistent with studies in other pLGIC and demonstrate the importance of the expression system used. Of the many differences between these two expression systems, we suggest that the different lipid content of the plasma membrane is a possible candidate for explaining these discrepancies.

Characterization of individual zinc binding sites in the cation diffusion facilitator YiiP

YiiP is a zinc/proton antiporter in the Cation Diffusion Facilitator superfamily. YiiP contributes to zinc homoeostasis by coupling zinc export to the proton motive force. Zinc binds at three sites: Site A in the transmembrane domain (TMD) constitutes the transport site. Site B is in a poorly conserved cytoplasmic intracellular loop connecting transmembrane helices M2 and M3. Site C binds two zinc ions in the cytoplasmic, C-terminal domain (CTD). Previous cryo-EM analysis revealed a novel conformational change featuring a kink between the TMD and the CTD, bending of M2 and M5 helices, and closure of a hydrophobic gate hypothesized to control the accessibility of the transport site.