Toward elucidating the heat activation mechanism of the TRPV1 channel gating by molecular dynamics simulation

As a key cellular sensor, the TRPV1 cation channel undergoes a gating transition from a closed state to an open state in response to various physical and chemical stimuli including noxious heat. Despite years of study, the heat activation mechanism of TRPV1 gating remains enigmatic at the molecular level. Toward elucidating the structural and energetic basis of TRPV1 gating, we have performed molecular dynamics (MD) simulations (with cumulative simulation time of 3 μs), starting from the high‐resolution closed and open structures of TRPV1 solved by cryo‐electron microscopy. In the closed‐state simulations at 30°C, we observed a stably closed channel constricted at the lower gate (near residue I679), while the upper gate (near residues G643 and M644) is dynamic and undergoes flickery opening/closing. In the open‐state simulations at 60°C, we found higher conformational variation consistent with a large entropy increase required for the heat activation, and both the lower and upper gates are dynamic with transient opening/closing. Through ensemble‐based structural analyses of the closed state versus the open state, we revealed pronounced closed‐to‐open conformational changes involving the membrane proximal domain (MPD) linker, the outer pore, and the TRP helix, which are accompanied by breaking/forming of a network of closed/open‐state specific hydrogen bonds. By comparing the closed‐state simulations at 30°C and 60°C, we observed heat‐activated conformational changes in the MPD linker, the outer pore, and the TRP helix that resemble the closed‐to‐open conformational changes, along with partial formation of the open‐state specific hydrogen bonds. Some of the residues involved in the above key hydrogen bonds were validated by previous mutational studies. Taken together, our MD simulations have offered rich structural and dynamic details beyond the static structures of TRPV1, and promising targets for future mutagenesis and functional studies of the TRPV1 channel. Proteins 2016; 84:1938–1949. © 2016 Wiley Periodicals, Inc.     

A yeast genetic screen reveals a critical role for the pore helix domain in TRP channel gating

TRP cation channels function as cellular sensors in uni- and multicellular eukaryotes. Despite intensive study, the mechanisms of TRP channel activation by chemical or physical stimuli remain poorly understood. To identify amino acid residues crucial for TRP channel gating, we developed an unbiased, high-throughput genetic screen in yeast that uncovered rare, constitutively active mutants of the capsaicin receptor, TRPV1. We show that mutations within the pore helix domain dramatically increase basal channel activity and responsiveness to chemical and thermal stimuli. Mutation of corresponding residues within two related TRPV channels leads to comparable effects on their activation properties. Our data suggest that conformational changes in the outer pore region are critical for determining the balance between open and closed states, providing evidence for a general role for this domain in TRP channel activation.     

The Integrity of the TRP Domain Is Pivotal for Correct TRPV1 Channel Gating

Transient receptor potential vanilloid subtype I (TRPV1) is a thermosensory ion channel that is also gated by chemical substances such as vanilloids. Adjacent to the channel gate, this polymodal thermoTRP channel displays a TRP domain, referred to as AD1, that plays a role in subunit association and channel gating. Previous studies have shown that swapping the AD1 in TRPV1 with the cognate from the TRPV2 channel (AD2) reduces protein expression and produces a nonfunctional chimeric channel (TRPV1-AD2). Here, we used a stepwise, sequential, cumulative site-directed mutagenesis approach, based on rebuilding the AD1 domain in the TRPV1-AD2 chimera, to unveil the minimum number of amino acids needed to restore protein expression and polymodal channel activity. Unexpectedly, we found that virtually full restitution of the AD1 sequence is required to reinstate channel expression and responses to capsaicin, temperature, and voltage. This strategy identified E692, R701, and T704 in the TRP domain as important for TRPV1 activity. Even conservative mutagenesis at these sites (E692D/R701K/T704S) impaired channel expression and abolished TRPV1 activity. However, the sole mutation of these positions in the TRPV1-AD2 chimera (D692E/K701R/S704T) was not sufficient to rescue channel gating, implying that other residues in the TRP domain are necessary to endow activity to TRPV1-AD2. A biophysical analysis of a functional chimera suggested that mutations in the TRP domain raised the energetics of channel gating by altering the coupling of stimuli sensing and pore opening. These findings indicate that inter- and/or intrasubunit interactions in the TRP domain are essential for correct TRPV1 gating.     

Toward decrypting the allosteric mechanism of the ryanodine receptor based on coarse‐grained structural and dynamic modeling

The ryanodine receptors (RyRs) are a family of calcium (Ca) channels that regulate Ca release by undergoing a closed‐to‐open gating transition in response to action potential or Ca binding. The allosteric mechanism of RyRs gating, which is activated/regulated by ligand/protein binding >200 Å away from the channel gate, remains elusive for the lack of high‐resolution structures. Recent solution of the closed‐form structures of the RyR1 isoform by cryo‐electron microscopy has paved the way for detailed structure‐driven studies of RyRs functions. Toward elucidating the allosteric mechanism of RyRs gating, we performed coarse‐grained modeling based on the newly solved closed‐form structures of RyR1. Our normal mode analysis captured a key mode of collective motions dominating the observed structural variations in RyR1, which features large outward and downward movements of the peripheral domains with the channel remaining closed, and involves hotspot residues that overlap well with key functional sites and disease mutations. In particular, we found a key interaction between a peripheral domain and the Ca‐binding EF hand domain, which may allow for direct coupling of Ca binding to the collective motions as captured by the above mode. This key mode was robustly reproduced by the normal mode analysis of the other two closed‐form structures of RyR1 solved independently. To elucidate the closed‐to‐open conformational changes in RyR1 with amino‐acid level of details, we flexibly fitted the closed‐form structures of RyR1 into a 10‐Å cryo‐electron microscopy map of the open state. We observed extensive structural changes involving the peripheral domains and the central domains, resulting in the channel pore opening. In sum, our findings have offered unprecedented structural and dynamic insights to the allosteric mechanism of RyR1 via modulation of the key collective motions involved in RyR1 gating. The predicted hotspot residues and open‐form conformation of RyR1 will guide future mutational and functional studies. Proteins 2015; 83:2307–2318. © 2015 Wiley Periodicals, Inc.     

Decrypting the Heat Activation Mechanism of TRPV1 Channel by Molecular Dynamics Simulation

As a prototype cellular sensor, the TRPV1 cation channel undergoes a closed-to-open gating transition in response to various physical and chemical stimuli including noxious heat. Despite recent progress, the molecular mechanism of heat activation of TRPV1 gating remains enigmatic. Toward decrypting the structural basis of TRPV1 heat activation, we performed extensive molecular dynamics simulations (with cumulative simulation time of ～11 μs) for the wild-type channel and a constitutively active double mutant at different temperatures (30, 60, and 72°C), starting from a high-resolution closed-channel structure of TRPV1 solved by cryo-electron microscopy. In the wild-type simulations, we observed heat-activated conformational changes (e.g., expansion or contraction) in various key domains of TRPV1 (e.g., the S2-S3 and S4-S5 linkers) to prime the channel for gating. These conformational changes involve a number of dynamic hydrogen-bond interactions that were validated with previous mutational studies. Next, our mutant simulations observed channel opening after a series of conformational changes that propagate from the channel periphery to the channel pore via key intermediate domains (including the S2-S3 and S4-S5 linkers). The gating transition is accompanied by a large increase in the protein-water electrostatic interaction energy, which supports the contribution of desolvation of polar/charged residues to the temperature-sensitive TRPV1 gating. Taken together, our molecular dynamics simulations and analyses offered, to our knowledge, new structural, dynamic, and energetic information to guide future mutagenesis and functional studies of the TRPV1 channels and development of TRPV1-targeting drugs.     

Mutation of I696 and W697 in the TRP box of vanilloid receptor subtype I modulates allosteric channel activation

The transient receptor potential vanilloid receptor subtype I (TRPV1) channel acts as a polymodal sensory receptor gated by chemical and physical stimuli. Like other TRP channels, TRPV1 contains in its C terminus a short, conserved domain called the TRP box, which is necessary for channel gating. Substitution of two TRP box residues—I696 and W697—with Ala markedly affects TRPV1’s response to all activating stimuli, which indicates that these two residues play a crucial role in channel gating. We systematically replaced I696 and W697 with 18 native l-amino acids (excluding cysteine) and evaluated the effect on voltage- and capsaicin-dependent gating. Mutation of I696 decreased channel activation by either voltage or capsaicin; furthermore, gating was only observed with substitution of hydrophobic amino acids. Substitution of W697 with any of the 18 amino acids abolished gating in response to depolarization alone, shifting the threshold to unreachable voltages, but not capsaicin-mediated gating. Moreover, vanilloid-activated responses of W697X mutants showed voltage-dependent gating along with a strong voltage-independent component. Analysis of the data using an allosteric model of activation indicates that mutation of I696 and W697 primarily affects the allosteric coupling constants of the ligand and voltage sensors to the channel pore. Together, our findings substantiate the notion that inter- and/or intrasubunit interactions at the level of the TRP box are critical for efficient coupling of stimulus sensing and gate opening. Perturbation of these interactions markedly reduces the efficacy and potency of the activating stimuli. Furthermore, our results identify these interactions as potential sites for pharmacological intervention.     

Comparative sequence analysis suggests a conserved gating mechanism for TRP channels

The transient receptor potential (TRP) channel superfamily plays a central role in transducing diverse sensory stimuli in eukaryotes. Although dissimilar in sequence and domain organization, all known TRP channels act as polymodal cellular sensors and form tetrameric assemblies similar to those of their distant relatives, the voltage-gated potassium (Kv) channels. Here, we investigated the related questions of whether the allosteric mechanism underlying polymodal gating is common to all TRP channels, and how this mechanism differs from that underpinning Kv channel voltage sensitivity. To provide insight into these questions, we performed comparative sequence analysis on large, comprehensive ensembles of TRP and Kv channel sequences, contextualizing the patterns of conservation and correlation observed in the TRP channel sequences in light of the well-studied Kv channels. We report sequence features that are specific to TRP channels and, based on insight from recent TRPV1 structures, we suggest a model of TRP channel gating that differs substantially from the one mediating voltage sensitivity in Kv channels. The common mechanism underlying polymodal gating involves the displacement of a defect in the H-bond network of S6 that changes the orientation of the pore-lining residues at the hydrophobic gate.     

Toxin binding reveals two open state structures for one acid-sensing ion channel

Of the three principal conformations of acid-sensing ion channels (ASICs)—closed, open and desensitized—only the atomic structure of the desensitized conformation had been known. Two recent papers report the crystal structure of chicken ASIC1 in complex with the spider toxin psalmotoxin 1, and one of these studies finds that, depending on the pH, channels are in two different open conformations. Compared with the desensitized conformation, toxin binding induces only subtle structural changes in the lower part of the large extracellular domain but a complete rearrangement of the two transmembrane domains (TMDs), suggesting that desensitization gating (the transition from open to desensitized) is mainly associated with conformational rearrangements of the TMDs. Moreover, the study reveals how two different arrangements of the TMDs in the open state give rise to ion pores with different selectivity for monovalent cations.     

Toxin binding reveals two open state structures for one acid-sensing ion channel

Of the three principal conformations of acid-sensing ion channels (ASICs)—closed, open and desensitized—only the atomic structure of the desensitized conformation had been known. Two recent papers report the crystal structure of chicken ASIC1 in complex with the spider toxin psalmotoxin 1, and one of these studies finds that, depending on the pH, channels are in two different open conformations. Compared with the desensitized conformation, toxin binding induces only subtle structural changes in the lower part of the large extracellular domain but a complete rearrangement of the two transmembrane domains (TMDs), suggesting that desensitization gating (the transition from open to desensitized) is mainly associated with conformational rearrangements of the TMDs. Moreover, the study reveals how two different arrangements of the TMDs in the open state give rise to ion pores with different selectivity for monovalent cations.     

Modulation of TRPV2 by endogenous and exogenous ligands: A computational study

Transient receptor potential vanilloid (TRPV) channels play various important roles in human physiology. As membrane proteins, these channels are modulated by their endogenous lipid environment as the recent wealth of structural studies has revealed functional and structural lipid binding sites. Additionally, it has been shown that exogenous ligands can exchange with some of these lipids to alter channel gating. Here, we used molecular dynamics simulations to examine how one member of the TRPV family, TRPV2, interacts with endogenous lipids and the pharmacological modulator cannabidiol (CBD). By computationally reconstituting TRPV2 into a typical plasma membrane environment, which includes phospholipids, cholesterol, and phosphatidylinositol (PIP) in the inner leaflet, we showed that most of the interacting surface lipids are phospholipids without strong specificity for headgroup types. Intriguingly, we observed that the C-terminal membrane proximal region of the channel binds preferentially to PIP lipids. We also modelled two structural lipids in the simulation: one in the vanilloid pocket and the other in the voltage sensor-like domain (VSLD) pocket. The simulation shows that the VSLD lipid dampens the fluctuation of the VSLD residues, while the vanilloid lipid exhibits heterogeneity both in its binding pose and in its influence on protein dynamics. Addition of CBD to our simulation system led to an open selectivity filter and a structural rearrangement that includes a clockwise rotation of the ankyrin repeat domains, TRP helix, and VSLD. Together, these results reveal the interplay between endogenous lipids and an exogenous ligand and their effect on TRPV2 stability and channel gating.     

Inter-Subunit Interactions across the Upper Voltage Sensing-Pore Domain Interface Contribute to the Concerted Pore Opening Transition of Kv Channels

The tight electro-mechanical coupling between the voltage-sensing and pore domains of Kv channels lies at the heart of their fundamental roles in electrical signaling. Structural data have identified two voltage sensor pore inter-domain interaction surfaces, thus providing a framework to explain the molecular basis for the tight coupling of these domains. While the contribution of the intra-subunit lower domain interface to the electro-mechanical coupling that underlies channel opening is relatively well understood, the contribution of the inter-subunit upper interface to channel gating is not yet clear. Relying on energy perturbation and thermodynamic coupling analyses of tandem-dimeric Shaker Kv channels, we show that mutation of upper interface residues from both sides of the voltage sensor-pore domain interface stabilizes the closed channel state. These mutations, however, do not affect slow inactivation gating. We, moreover, find that upper interface residues form a network of state-dependent interactions that stabilize the open channel state. Finally, we note that the observed residue interaction network does not change during slow inactivation gating. The upper voltage sensing-pore interaction surface thus only undergoes conformational rearrangements during channel activation gating. We suggest that inter-subunit interactions across the upper domain interface mediate allosteric communication between channel subunits that contributes to the concerted nature of the late pore opening transition of Kv channels.     

The S4–­­S5 linker – gearbox of TRP channel gating

Transient receptor potential (TRP) channels are cation channels which participate in a wide variety of physiological processes in organisms ranging from fungi to humans. They fulfill roles in body homeostasis, are sensors for noxious chemicals and temperature in the mammalian somatosensory system and are activated by light stimulated phospholipase C activity in Drosophila or by hypertonicity in yeast. The transmembrane topology of TRP channels is similar to that of voltage-gated cation channels. TRP proteins assemble as tetramers with each subunit containing six transmembrane helices (S1–S6) and intracellular N- and C-termini. Here we focus on the emerging functions of the cytosolic S4–S5 linker on TRP channel gating. Most of this knowledge comes from pathogenic mutations within the S4–S5 linker that alter TRP channel activities. This knowledge has stimulated forward genetic approaches to identify additional residues around this region which are essential for channel gating and is supported, in part, by recent structures obtained for TRPV1, TRPV2, TRPV6, TRPA1, and TRPP2.     

Conserved residues within the putative S4-S5 region serve distinct functions among thermosensitive vanilloid transient receptor potential (TRPV) channels

The vanilloid transient receptor potential channel TRPV1 is a tetrameric six-transmembrane segment (S1-S6) channel that can be synergistically activated by various proalgesic agents such as capsaicin, protons, heat, or highly depolarizing voltages, and also by 2-aminoethoxydiphenyl borate (2-APB), a common activator of the related thermally gated vanilloid TRP channels TRPV1, TRPV2, and TRPV3. In these channels, the conserved charged residues in the intracellular S4-S5 region have been proposed to constitute part of a voltage sensor that acts in concert with other stimuli to regulate channel activation. The molecular basis of this gating event is poorly understood. We mutated charged residues all along the S4 and the S4-S5 linker of TRPV1 and identified four potential voltage-sensing residues (Arg(557), Glu(570), Asp(576), and Arg(579)) that, when specifically mutated, altered the functionality of the channel with respect to voltage, capsaicin, heat, 2-APB, and/or their interactions in different ways. The nonfunctional charge-reversing mutations R557E and R579E were partially rescued by the charge-swapping mutations R557E/E570R and D576R/R579E, indicating that electrostatic interactions contribute to allosteric coupling between the voltage-, temperature- and capsaicin-dependent activation mechanisms. The mutant K571E was normal in all aspects of TRPV1 activation except for 2-APB, revealing the specific role of Lys(571) in chemical sensitivity. Surprisingly, substitutions at homologous residues in TRPV2 or TRPV3 had no effect on temperature- and 2-APB-induced activity. Thus, the charged residues in S4 and the S4-S5 linker contribute to voltage sensing in TRPV1 and, despite their highly conserved nature, regulate the temperature and chemical gating in the various TRPV channels in different ways.     

Citral sensing by Transient [corrected] receptor potential channels in dorsal root ganglion neurons

Transient receptor potential (TRP) ion channels mediate key aspects of taste, smell, pain, temperature sensation, and pheromone detection. To deepen our understanding of TRP channel physiology, we require more diverse pharmacological tools. Citral, a bioactive component of lemongrass, is commonly used as a taste enhancer, as an odorant in perfumes, and as an insect repellent. Here we report that citral activates TRP channels found in sensory neurons (TRPV1 and TRPV3, TRPM8, and TRPA1), and produces long-lasting inhibition of TRPV1-3 and TRPM8, while transiently blocking TRPV4 and TRPA1. Sustained citral inhibition is independent of internal calcium concentration, but is state-dependent, developing only after TRP channel opening. Citral's actions as a partial agonist are not due to cysteine modification of the channels nor are they a consequence of citral's stereoisoforms. The isolated aldehyde and alcohol cis and trans enantiomers (neral, nerol, geranial, and geraniol) each reproduce citral's actions. In juvenile rat dorsal root ganglion neurons, prolonged citral inhibition of native TRPV1 channels enabled the separation of TRPV2 and TRPV3 currents. We find that TRPV2 and TRPV3 channels are present in a high proportion of these neurons (94% respond to 2-aminoethyldiphenyl borate), consistent with our immunolabeling experiments and previous in situ hybridization studies. The TRPV1 activation requires residues in transmembrane segments two through four of the voltage-sensor domain, a region previously implicated in capsaicin activation of TRPV1 and analogous menthol activation of TRPM8. Citral's broad spectrum and prolonged sensory inhibition may prove more useful than capsaicin for allodynia, itch, or other types of pain involving superficial sensory nerves and skin.     

Mutations in the S4 region isolate the final voltage-dependent cooperative step in potassium channel activation

Charged residues in the S4 transmembrane segment play a key role in determining the sensitivity of voltage-gated ion channels to changes in voltage across the cell membrane. However, cooperative interactions between subunits also affect the voltage dependence of channel opening, and these interactions can be altered by making substitutions at uncharged residues in the S4 region. We have studied the activation of two mutant Shaker channels that have different S4 amino acid sequences, ILT (V369I, I372L, and S376T) and Shaw S4 (the S4 of Drosophila Shaw substituted into Shaker), and yet have very similar ionic current properties. Both mutations affect cooperativity, making a cooperative transition in the activation pathway rate limiting and shifting it to very positive voltages, but analysis of gating and ionic current recordings reveals that the ILT and Shaw S4 mutant channels have different activation pathways. Analysis of gating currents suggests that the dominant effect of the ILT mutation is to make the final cooperative transition to the open state of the channel rate limiting in an activation pathway that otherwise resembles that of Shaker. The charge movement associated with the final gating transition in ILT activation can be measured as an isolated component of charge movement in the voltage range of channel opening and accounts for 13% ( approximately 1.8 e0) of the total charge moved in the ILT activation pathway. The remainder of the ILT gating charge (87%) moves at negative voltages, where channels do not open, and confirms the presence of Shaker-like conformational changes between closed states in the activation pathway. In contrast to ILT, the activation pathway of Shaw S4 seems to involve a single cooperative charge-moving step between a closed and an open state. We cannot detect any voltage-dependent transitions between closed states for Shaw S4. Restoring basic residues that are missing in Shaw S4 (R1, R2, and K7) rescues charge movement between closed states in the activation pathway, but does not alter the voltage dependence of the rate-limiting transition in activation.     

Gating Transition of Pentameric Ligand-Gated Ion Channels

Pentameric ligand-gated ion channels are an important family of membrane proteins and play key roles in physiological processes, including signal transduction at chemical synapses. Here, we study the conformational changes associated with the opening and closing of the channel pore. Based on recent crystal structures of two prokaryotic members of the family in open and closed states, respectively, mixed elastic network models are constructed for the transmembrane domain. To explore the conformational changes in the gating transition, a coarse-grained transition path is computed that smoothly connects the closed and open conformations of the channel. We find that the conformational transition involves no major rotations of the transmembrane helices, and is instead characterized by a concerted tilting of helices M2 and M3. In addition, helix M2 changes its bending state, which results in an early closure of the pore during the open-to-closed transition.     

A conserved gating element in TRPV6 channels

The Ca²⁺-selective tetrameric Transient Receptor Potential Vanilloid 6 (TRPV6) channel is an inwardly rectifying ion channel. The constitutive current endures Ca²⁺-induced inactivation as a result of the activation of phospholipase C followed depletion of phosphatidylinositol 4,5-bisphosphate, and calmodulin binding. Replacing a glycine residue within the cytosolic S4-S5 linker of the human TRPV6 protein, glycine 516, which is conserved in all TRP channel proteins, by a serine residue forces the channels into an open conformation thereby enhancing constitutive Ca²⁺ entry and preventing inactivation. Introduction of a second mutation (T621A) into TRPV6_G516S reduces constitutive activity and partially rescues the TRPV6 function. According to the recently revealed crystal structure of the rat TRPV6 the T621 is adjacent to the distal end of the transmembrane segment 6 (S6) within a short linker between S6 and the helix formed by the TRP domain. These results indicate that the S4-S5 linker and the S6-TRP-domain linker are critical constituents of TRPV6 channel gating and that disturbance of their sequences foster constitutive Ca²⁺ entry.     

Citral Sensing by TRANSient Receptor Potential Channels in Dorsal Root Ganglion Neurons

Transient receptor potential (TRP) ion channels mediate key aspects of taste, smell, pain, temperature sensation, and pheromone detection. To deepen our understanding of TRP channel physiology, we require more diverse pharmacological tools. Citral, a bioactive component of lemongrass, is commonly used as a taste enhancer, as an odorant in perfumes, and as an insect repellent. Here we report that citral activates TRP channels found in sensory neurons (TRPV1 and TRPV3, TRPM8, and TRPA1), and produces long-lasting inhibition of TRPV1–3 and TRPM8, while transiently blocking TRPV4 and TRPA1. Sustained citral inhibition is independent of internal calcium concentration, but is state-dependent, developing only after TRP channel opening. Citral''s actions as a partial agonist are not due to cysteine modification of the channels nor are they a consequence of citral''s stereoisoforms. The isolated aldehyde and alcohol cis and trans enantiomers (neral, nerol, geranial, and geraniol) each reproduce citral''s actions. In juvenile rat dorsal root ganglion neurons, prolonged citral inhibition of native TRPV1 channels enabled the separation of TRPV2 and TRPV3 currents. We find that TRPV2 and TRPV3 channels are present in a high proportion of these neurons (94% respond to 2-aminoethyldiphenyl borate), consistent with our immunolabeling experiments and previous in situ hybridization studies. The TRPV1 activation requires residues in transmembrane segments two through four of the voltage-sensor domain, a region previously implicated in capsaicin activation of TRPV1 and analogous menthol activation of TRPM8. Citral''s broad spectrum and prolonged sensory inhibition may prove more useful than capsaicin for allodynia, itch, or other types of pain involving superficial sensory nerves and skin.     

Hinge-bending motions in the pore domain of a bacterial voltage-gated sodium channel

Computational methods and experimental data are used to provide structural models for NaChBac, the homo-tetrameric voltage-gated sodium channel from the bacterium Bacillus halodurans, with a closed and partially open pore domain. Molecular dynamic (MD) simulations on membrane-bound homo-tetrameric NaChBac structures, each comprising six helical transmembrane segments (labeled S1 through S6), reveal that the shape of the lumen, which is defined by the bundle of four alpha-helical S6 segments, is modulated by hinge bending motions around the S6 glycine residues. Mutation of these glycine residues into proline and alanine affects, respectively, the structure and conformational flexibility of the S6 bundle. In the closed channel conformation, a cluster of stacked phenylalanine residues from the four S6 helices hinders diffusion of water molecules and Na(+) ions. Activation of the voltage sensor domains causes destabilization of the aforementioned cluster of phenylalanines, leading to a more open structure. The conformational change involving the phenylalanine cluster promotes a kink in S6, suggesting that channel gating likely results from the combined action of hinge-bending motions of the S6 bundle and concerted reorientation of the aromatic phenylalanine side-chains.     

An extracellular domain of the accessory β1 subunit is required for modulating BK channel voltage sensor and gate

A family of tissue-specific auxiliary β subunits modulates large conductance voltage- and calcium-activated potassium (BK) channel gating properties to suit their diverse functions. Paradoxically, β subunits both promote BK channel activation through a stabilization of voltage sensor activation and reduce BK channel openings through an increased energetic barrier of the closed-to-open transition. The molecular determinants underlying β subunit function, including the dual gating effects, remain unknown. In this study, we report the first identification of a β1 functional domain consisting of Y74, S104, Y105, and I106 residues located in the extracellular loop of β1. These amino acids reside within two regions of highest conservation among related β1, β2, and β4 subunits. Analysis in the context of the Horrigan-Aldrich gating model revealed that this domain functions to both promote voltage sensor activation and also reduce intrinsic gating. Free energy calculations suggest that the dual effects of the β1 Y74 and S104-I106 domains can be largely accounted for by a relative destabilization of channels in open states that have few voltage sensors activated. These results suggest a unique and novel mechanism for β subunit modulation of voltage-gated potassium channels wherein interactions between extracellular β subunit residues with the external portions of the gate and voltage sensor regulate channel opening.