S4-S5 Linker Research Articles

Insights into the molecular mechanism for hyperpolarization-dependent activation of HCN channels

Hyperpolarization-activated, cyclic nucleotide-gated (HCN) ion channels are both voltage- and ligand-activated membrane proteins that contribute to electrical excitability and pace-making activity in cardiac and neuronal cells. These channels are members of the voltage-gated Kv channel superfamily and cyclic nucleotide-binding domain subfamily of ion channels. HCN channels have a unique feature that distinguishes them from other voltage-gated channels: the HCN channel pore opens in response to hyperpolarizing voltages instead of depolarizing voltages. In the canonical model of electromechanical coupling, based on Kv channels, a change in membrane voltage activates the voltage-sensing domains (VSD) and the activation energy passes to the pore domain (PD) through a covalent linker that connects the VSD to the PD. In this investigation, the covalent linkage between the VSD and PD, the S4-S5 linker, and nearby regions of spHCN channels were mutated to determine the functional role each plays in hyperpolarization-dependent activation. The results show that: (i) the S4-S5 linker is not required for hyperpolarization-dependent activation or ligand-dependent gating; (ii) the S4 C-terminal region (S4C-term) is not necessary for ligand-dependent gating but is required for hyperpolarization-dependent activation and acts like an autoinhibitory domain on the PD; (iii) the S5N-term region is involved in VSD-PD coupling and holding the pore closed; and (iv) spHCN channels have two voltage-dependent processes, a hyperpolarization-dependent activation and a depolarization-dependent recovery from inactivation. These results are inconsistent with the canonical model of VSD-PD coupling in Kv channels and elucidate the mechanism for hyperpolarization-dependent activation of HCN channels.

The conformational wave in capsaicin activation of transient receptor potential vanilloid 1 ion channel

The capsaicin receptor TRPV1 has been intensively studied by cryo-electron microscopy and functional tests. However, though the apo and capsaicin-bound structural models are available, the dynamic process of capsaicin activation remains intangible, largely due to the lack of a capsaicin-induced open structural model and the low occupancy of the transition states. Here we report that reducing temperature toward the freezing point substantially increased channel closure events even in the presence of saturating capsaicin. We further used a combination of fluorescent unnatural amino acid (fUAA) incorporation, computational modeling, and rate-equilibrium linear free-energy relationships analysis (Φ-analysis) to derive the fully open capsaicin-bound state model, and reveal how the channel transits from the apo to the open state. We observed that capsaicin initiates a conformational wave that propagates through the S4–S5 linker towards the S6 bundle and finally reaching the selectivity filter. Our study provides a temporal mechanism for capsaicin activation of TRPV1.

Propofol inhibits prokaryotic voltage-gated Na+ channels by promoting activation-coupled inactivation.

Propofol is widely used in the clinic for the induction and maintenance of general anesthesia. As with most general anesthetics, however, our understanding of its mechanism of action remains incomplete. Local and general anesthetics largely inhibit voltage-gated Na+ channels (Navs) by inducing an apparent stabilization of the inactivated state, associated in some instances with pore block. To determine the biophysical and molecular basis of propofol action in Navs, we investigated NaChBac and NavMs, two prokaryotic Navs with distinct voltage dependencies and gating kinetics, by whole-cell patch clamp electrophysiology in the absence and presence of propofol at clinically relevant concentrations (2-10 µM). In both Navs, propofol induced a hyperpolarizing shift of the pre-pulse inactivation curve without any significant effects on recovery from inactivation at strongly hyperpolarized voltages, demonstrating that propofol does not stabilize the inactivated state. Moreover, there was no evidence of fast or slow pore block by propofol in a non-inactivating NaChBac mutant (T220A). Propofol also induced hyperpolarizing shifts of the conductance-voltage relationships with negligible effects on the time constants of deactivation at hyperpolarized voltages, indicating that propofol does not stabilize the open state. Instead, propofol decreases the time constants of macroscopic activation and inactivation. Adopting a kinetic scheme of Nav gating that assumes preferential closed-state recovery from inactivation, a 1.7-fold acceleration of the rate constant of activation and a 1.4-fold acceleration of the rate constant of inactivation were sufficient to reproduce experimental observations with computer simulations. In addition, molecular dynamics simulations and molecular docking suggest that propofol binding involves interactions with gating machinery in the S4-S5 linker and external pore regions. Our findings show that propofol is primarily a positive gating modulator of prokaryotic Navs, which ultimately inhibits the channels by promoting activation-coupled inactivation.

Propofol inhibits the voltage-gated sodium channel NaChBac at multiple sites.

Voltage-gated sodium (NaV) channels are important targets of general anesthetics, including the intravenous anesthetic propofol. Electrophysiology studies on the prokaryotic NaV channel NaChBac have demonstrated that propofol promotes channel activation and accelerates activation-coupled inactivation, but the molecular mechanisms of these effects are unclear. Here, guided by computational docking and molecular dynamics simulations, we predict several propofol-binding sites in NaChBac. We then strategically place small fluorinated probes at these putative binding sites and experimentally quantify the interaction strengths with a fluorinated propofol analogue, 4-fluoropropofol. In vitro and in vivo measurements show that 4-fluoropropofol and propofol have similar effects on NaChBac function and nearly identical anesthetizing effects on tadpole mobility. Using quantitative analysis by 19F-NMR saturation transfer difference spectroscopy, we reveal strong intermolecular cross-relaxation rate constants between 4-fluoropropofol and four different regions of NaChBac, including the activation gate and selectivity filter in the pore, the voltage sensing domain, and the S4-S5 linker. Unlike volatile anesthetics, 4-fluoropropofol does not bind to the extracellular interface of the pore domain. Collectively, our results show that propofol inhibits NaChBac at multiple sites, likely with distinct modes of action. This study provides a molecular basis for understanding the net inhibitory action of propofol on NaV channels.

S4–S5 linker movement during activation and inactivation in voltage-gated K + channels

The S4-S5 linker physically links voltage sensor and pore domain in voltage-gated ion channels and is essential for electromechanical coupling between both domains. Little dynamic information is available on the movement of the cytosolic S4-S5 linker due to lack of a direct electrical or optical readout. To understand the movements of the gating machinery during activation and inactivation, we incorporated fluorescent unnatural amino acids at four positions along the linker of the Shaker KV channel. Using two-color voltage-clamp fluorometry, we compared S4-S5 linker movements with charge displacement, S4 movement, and pore opening. We found that the proximal S4-S5 linker moves with the S4 helix throughout the gating process, whereas the distal portion undergoes a separate motion related to late gating transitions. Both pore and S4-S5 linker undergo rearrangements during C-type inactivation. In presence of accelerated C-type inactivation, the energetic coupling between movement of the distal S4-S5 linker and pore opening disappears.

Mouse β-Defensin 3, A Defensin Inhibitor of Both Its Endogenous and Exogenous Potassium Channels.

The human defensins are recently discovered to inhibit potassium channels, which are classical targets of the animal toxins. Whether other vertebrate defensins are potassium channel inhibitors remains unknown. In this work, we reported that the mouse β-defensin 3 (mBD3) was a novel inhibitor of both endogenous and exogenous potassium channels. The structural analysis showed that mBD3 is the most identical to human Kv1.3 channel-sensitive human β-defensin 2 (hBD2). However, the pharmacological profiles indicated that the recombinant mBD3 (rmBD3) weakly inhibited the mouse and human Kv1.3 channels. Different from the pharmacological features of human β-defensins, mBD3 more selectively inhibited the mouse Kv1.6 and human KCNQ1/KCNE1 channels with IC50 values of 0.6 ± 0.4 μM and 1.2 ± 0.8 μM, respectively. The site directed mutagenesis experiments indicated that the extracellular pore region of mouse Kv1.6 channel was the interaction site of rmBD3. In addition, the minor effect on the channel conductance-voltage relationship curves implied that mBD3 might bind the extracellular transmembrane helices S1-S2 linker and/or S3-S4 linker of mouse Kv1.6 channel. Together, these findings not only revealed mBD3 as a novel inhibitor of both endogenous and exogenous potassium channels, but also provided a clue to investigate the role of mBD3-Kv1.6 channel interaction in the physiological and pathological field in the future.

Structure of the human lipid-gated cation channel TRPC3.

The TRPC channels are crucially involved in store-operated calcium entry and calcium homeostasis, and they are implicated in human diseases such as neurodegenerative disease, cardiac hypertrophy, and spinocerebellar ataxia. We present a structure of the full-length human TRPC3, a lipid-gated TRPC member, in a lipid-occupied, closed state at 3.3 Angstrom. TRPC3 has four elbow-like membrane reentrant helices prior to the first transmembrane helix. The TRP helix is perpendicular to, and thus disengaged from, the pore-lining S6, suggesting a different gating mechanism from other TRP subfamily channels. The third transmembrane helix S3 is remarkably long, shaping a unique transmembrane domain, and constituting an extracellular domain that may serve as a sensor of external stimuli. We identified two lipid-binding sites, one being sandwiched between the pre-S1 elbow and the S4-S5 linker, and the other being close to the ion-conducting pore, where the conserved LWF motif of the TRPC family is located.

A Mixed Periodic Paralysis & Myotonia Mutant, P1158S, Imparts pH-Sensitivity in Skeletal Muscle Voltage-gated Sodium Channels

Skeletal muscle channelopathies, many of which are inherited as autosomal dominant mutations, include myotonia and periodic paralysis. Myotonia is defined by a delayed relaxation after muscular contraction, whereas periodic paralysis is defined by episodic attacks of weakness. One sub-type of periodic paralysis, known as hypokalemic periodic paralysis (hypoPP), is associated with low potassium levels. Interestingly, the P1158S missense mutant, located in the third domain S4-S5 linker of the “skeletal muscle”, Nav1.4, has been implicated in causing both myotonia and hypoPP. A common trigger for these conditions is physical activity. We previously reported that Nav1.4 is relatively insensitive to changes in extracellular pH compared to Nav1.2 and Nav1.5. Given that intense exercise is often accompanied by blood acidosis, we decided to test whether changes in pH would push gating in P1158S towards either phenotype. Our results suggest that, unlike in WT-Nav1.4, low pH depolarizes the voltage-dependence of activation and steady-state fast inactivation, decreases current density, and increases late currents in P1185S. Thus, P1185S turns the normally pH-insensitive Nav1.4 into a proton-sensitive channel. Using action potential modeling we predict a pH-to-phenotype correlation in patients with P1158S. We conclude that activities which alter blood pH may trigger the noted phenotypes in P1158S patients.

The regulatory mechanism of mammalian TRPMLs revealed by cryo-EM.

Transient receptor potential mucolipin (TRPML) channels are the most recently identified subfamily of TRP channels and have seen a surge of new reports revealing both structural and functional insight. In 2017, several groups published multiple conformations of TRPML channels using cryo-EM. Similar to other TRP channels, the ML subfamily consists of six transmembrane helices (S1-S6), and a pore region including S5, S6, and two pore helices (PH1 and PH2). However, these reports also reveal distinct structural characteristics of the ML subfamily. Asp residues within the luminal pore may function to control calcium/pH regulation. A synthetic agonist, ML-SA1, can bind to the pore region of TRPMLs to force a direct dilation of the lower gate. Finally, biophysical and electrophysiological characterizations reveal another natural agonist binding site in the unique domain of TRPMLs, presumably regulating the conformation of the S4-S5 linker to open the channel. This work elucidates the molecular architecture and provides insights into how multiple ligands regulate TRPMLs.

Gating interaction maps reveal a noncanonical electromechanical coupling mode in the Shaker K+ channel

Membrane potential regulates the activity of voltage-dependent ion channels via specialized voltage-sensing modules but the mechanisms involved in coupling voltage-sensor movement to pore opening remain unclear due to lack of resting state structures and robust methods to identify allosteric pathways. Here, using a newly developed interaction energy analysis, we probe the interfaces of the voltage-sensing and pore modules in the drosophila Shaker K+ channel. Our measurements reveal unexpectedly strong equilibrium gating interactions between contacts at the S4 and S5 helices in addition to those between S6 and S4–S5 linker. Network analysis of MD trajectories shows that the voltage-sensor and pore motions are linked by two distinct pathways- canonical one through the S4–S5 linker and a hitherto unknown pathway akin to rack and pinion coupling involving S4 and S5 helices. Our findings highlight the central role of the S5 helix in electromechanical transduction in the VGIC superfamily.

Contribution of S4 segments and S4-S5 linkers to the low-voltage activation properties of T-type CaV3.3 channels.

Voltage-gated calcium channels contain four highly conserved transmembrane helices known as S4 segments that exhibit a positively charged residue every third position, and play the role of voltage sensing. Nonetheless, the activation range between high-voltage (HVA) and low-voltage (LVA) activated calcium channels is around 30–40 mV apart, despite the high level of amino acid similarity within their S4 segments. To investigate the contribution of S4 voltage sensors for the low-voltage activation characteristics of CaV3.3 channels we constructed chimeras by swapping S4 segments between this LVA channel and the HVA CaV1.2 channel. The substitution of S4 segment of Domain II in CaV3.3 by that of CaV1.2 (chimera IIS4C) induced a ~35 mV shift in the voltage-dependence of activation towards positive potentials, showing an I-V curve that almost overlaps with that of CaV1.2 channel. This HVA behavior induced by IIS4C chimera was accompanied by a 2-fold decrease in the voltage-dependence of channel gating. The IVS4 segment had also a strong effect in the voltage sensing of activation, while substitution of segments IS4 and IIIS4 moved the activation curve of CaV3.3 to more negative potentials. Swapping of IIS4 voltage sensor influenced additional properties of this channel such as steady-state inactivation, current decay, and deactivation. Notably, Domain I voltage sensor played a major role in preventing CaV3.3 channels to inactivate from closed states at extreme hyperpolarized potentials. Finally, site-directed mutagenesis in the CaV3.3 channel revealed a partial contribution of the S4-S5 linker of Domain II to LVA behavior, with synergic effects observed in double and triple mutations. These findings indicate that IIS4 and, to a lesser degree IVS4, voltage sensors are crucial in determining the LVA properties of CaV3.3 channels, although the accomplishment of this function involves the participation of other structural elements like S4-S5 linkers.

Structure of full-length human TRPM4

Transient receptor potential melastatin subfamily member 4 (TRPM4) is a widely distributed, calcium-activated, monovalent-selective cation channel. Mutations in human TRPM4 (hTRPM4) result in progressive familial heart block. Here, we report the electron cryomicroscopy structure of hTRPM4 in a closed, Na+-bound, apo state at pH 7.5 to an overall resolution of 3.7 Å. Five partially hydrated sodium ions are proposed to occupy the center of the conduction pore and the entrance to the coiled-coil domain. We identify an upper gate in the selectivity filter and a lower gate at the entrance to the cytoplasmic coiled-coil domain. Intramolecular interactions exist between the TRP domain and the S4-S5 linker, N-terminal domain, and N and C termini. Finally, we identify aromatic interactions via π-π bonds and cation-π bonds, glycosylation at an N-linked extracellular site, a pore-loop disulfide bond, and 24 lipid binding sites. We compare and contrast this structure with other TRP channels and discuss potential mechanisms of regulation and gating of human full-length TRPM4.

A Non-canonical VSD-Pore Coupling is Responsible for the AO State of KCNQ1 Channels

In voltage dependent activation of Kv channels, membrane depolarization activates the voltage sensor(VSD), which then opens the pore via the VSD-pore coupling. The interaction between the S4-S5 linker and the cytosolic end of S6 has been proposed to mediate the VSD-pore coupling. KCNQ1 (Kv7.1) in association with the auxiliary subunit KCNE1 forms the IKs channel in the heart that is critical for controlling heart rhythm. It has been shown that KCNQ1 opens to two open states when the voltage sensor is activated to the intermediate state (termed IO) and the fully activated state (AO), respectively. The association of KCNE1 suppresses the IO state and enhances the AO state. Therefore, the IKschannel only opens to the AO state, which is more important to the cardiac physiology. We have previously shown that the F351A mutation selectively suppressed the IO state by disrupting the VSD-pore coupling. Since F351 is a conserved residue important for the above mentioned VSD-pore coupling mechanism, this result suggests that the canonical coupling mechanism is important for the IO state, In this study we find that three mutations of KCNQ1 that are associated with Long QT syndrome, S338F, F339S and L342A disrupt the VSD-pore coupling to selectively eliminate the AO state. These residues are located in the middle of S6 and cannot interact with the S4-S5 linker. These results indicate that different VSD-pore coupling mechanisms are responsible for the IO and AO states, and mutations in different structural motifs can selectively disrupt either of the mechanisms. Furthermore, the middle of S6 is involved in a non-canonical mechanism that specifically couples VSD activation to the AO state.

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 (MD) simulations (with cumulative simulation time of ∼11 μs) for the wildtype (WT) channel and a constitutively active double mutant at different temperatures (30°C, 60°C, and 72°C), starting from a high-resolution closed-channel structure of TRPV1 solved by cryo-electron microscopy (cryo-EM). In the WT 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 which were validated with previous mutational studies. Next, our mutant simulations observed channel opening following 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 de-solvation of polar/charged residues to the temperature-sensitive TRPV1 gating. Taken together, our MD simulations and analyses offered new structural, dynamic, and energetic information to guide future mutagenesis and functional studies of TRPV1 channel and development of TRPV1-targeting drugs.

Role of Hydrophobic Solvation in TRPV1 Temperature Sensitivity

TRPV1, a channel permeable to Ca2+, plays crucial role in pain sensation. The molecular details of TRPV1 gating, including its temperature dependence, remain largely unknown. Here we use a combined computational and experimental approach to shed light on the TRPV1 closed-to-open transition. We find that gating relies on the rotation of N676, an evolutionarily conserved residue on the S6 helix: N676 can either face the channel pore or the S4-S5 linker. Only in the former case, the channel pore is hydrated. When N676 rotates toward the linker, we observe hydration of four so far unreported cavities. Based on our findings, we propose a model for TRPV1 gating involving dynamic hydration of these cavities. Free energy calculations indicate that this gating mechanism is markedly temperature dependent. On the basis of this model, which rationalizes seemingly conflicting experimental observations, we predict and experimentally confirm the behavior of two mutants.

Alpha-B Helix of the RCK1 Domain is a Shared Structural Element for Both Voltage and Calcium Activation of BK Channels

BK channels are comprised of an integral-membrane core, that includes four voltage sensors and a central pore-gate domain (PGD), and also a large cytosolic domain (CTD), termed the gating ring, which is attached to the core through four S6-RCK1 peptide linkers. Structural studies (Yuan et al. 2011; Hite, Tao, MacKinnon, 2016) indicate that Ca2+ binding to the CTD rotates the N-lobe of each RCK1 outwards, which moves the α-B helices outwards and upwards to push on the core near the S4-S5 linkers, while simultaneously pulling the C-terminus of the S6-RCK1 linkers outwards and downwards. This gives a push-pull action between gating ring and core. We now further examine the contribution of the α-B helix to channel activation by introducing point proline mutations along the α-B helix. Such mutations right shifted V0.5 for voltage activation by +50 to +180 mV, while having negligible effects on the gating currents, indicating that the α-B helix is involved in voltage activation. The mutations also decreased the delta leftward shift in V0.5 due to Ca2+ activation, and this was also the case for the Ca2+ bowl and RCK1 sites acting separately, indicating that both high affinity Ca2+ binding sites act, at least in part, through the α-B helix. Higher Ca2+ did not restore the delta Ca2+ shift to that of WT. The crystal structure of the CTD with L390P indicated one less helical turn in the α-B helix than WT without other notable differences in the CTD. These findings indicate that an intact α-B helix is required for effective coupling of both Ca2+ binding and voltage depolarization to pore opening, and that a shared pathway involving the α-B helix may be involved. NIIH GM114694.

Distinct modulation of inactivation by a residue in the pore domain of voltage-gated Na+ channels: mechanistic insights from recent crystal structures

Inactivation of voltage-gated Na+ channels (VGSC) is essential for the regulation of cellular excitability. The molecular rearrangement underlying inactivation is thought to involve the intracellular linker between domains III and IV serving as inactivation lid, the receptor for the lid (domain III S4-S5 linker) and the pore-lining S6 segements. To better understand the role of the domain IV S6 segment in inactivation we performed a cysteine scanning mutagenesis of this region in rNav 1.4 channels and screened the constructs for perturbations in the voltage-dependence of steady state inactivation. This screen was performed in the background of wild-type channels and in channels carrying the mutation K1237E, which profoundly alters both permeation and gating-properties. Of all tested constructs the mutation I1581C was unique in that the mutation-induced gating changes were strongly influenced by the mutational background. This suggests that I1581 is involved in specific short-range interactions during inactivation. In recently published crystal structures VGSCs the respective amino acids homologous to I1581 appear to control a bend of the S6 segment which is critical to the gating process. Furthermore, I1581 may be involved in the transmission of the movement of the DIII voltage-sensor to the domain IV S6 segment.

Nonsensing residues in S3-S4 linker's C terminus affect the voltage sensor set point in K+ channels.

Voltage sensitivity in ion channels is a function of highly conserved arginine residues in their voltage-sensing domains (VSDs), but this conservation does not explain the diversity in voltage dependence among different K+ channels. Here we study the non-voltage-sensing residues 353 to 361 in Shaker K+ channels and find that residues 358 and 361 strongly modulate the voltage dependence of the channel. We mutate these two residues into all possible remaining amino acids (AAs) and obtain Q-V and G-V curves. We introduced the nonconducting W434F mutation to record sensing currents in all mutants except L361R, which requires K+ depletion because it is affected by W434F. By fitting Q-Vs with a sequential three-state model for two voltage dependence-related parameters (V0, the voltage-dependent transition from the resting to intermediate state and V1, from the latter to the active state) and G-Vs with a two-state model for the voltage dependence of the pore domain parameter (V1/2), Spearman's coefficients denoting variable relationships with hydrophobicity, available area, length, width, and volume of the AAs in 358 and 361 positions could be calculated. We find that mutations in residue 358 shift Q-Vs and G-Vs along the voltage axis by affecting V0, V1, and V1/2 according to the hydrophobicity of the AA. Mutations in residue 361 also shift both curves, but V0 is affected by the hydrophobicity of the AA in position 361, whereas V1 and V1/2 are affected by size-related AA indices. Small-to-tiny AAs have opposite effects on V1 and V1/2 in position 358 compared with 361. We hypothesize possible coordination points in the protein that residues 358 and 361 would temporarily and differently interact with in an intermediate state of VSD activation. Our data contribute to the accumulating knowledge of voltage-dependent ion channel activation by adding functional information about the effects of so-called non-voltage-sensing residues on VSD dynamics.

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.

Biophysical Characterization of Genetically Encoded Voltage Sensor ASAP1: Dynamic Range Improvement

Recent work has introduced a new fluorescent voltage sensor, ASAP1, which can monitor rapid trains of action potentials in cultured neurons. This indicator is based on the Gallus gallus voltage-sensitive phosphatase with the phosphatase domain removed and a circularly permuted GFP placed in the S3-S4 linker. However, many of the biophysical details of this indicator remain unknown. In this work, we study the biophysical properties of ASAP1. Using the cut-open voltage clamp technique, we have simultaneously recorded fluorescence signals and gating currents from Xenopus laevis oocytes expressing ASAP1. Gating charge movement and fluorescence kinetics track closely with each other, although ASAP1 gating currents are significantly faster than those of Ciona intestinalis voltage-sensitive phosphatase. Altering the residue before the first gating charge removes a split in the ASAP1 QV curve, but preserves the accelerated kinetics that allow for the faithful tracking of action potentials in neurons.