Voltage-sensing Domains Activation Research Articles

A binding site for phosphoinositides described by multiscale simulations explains their modulation of voltage-gated sodium channels

Voltage-gated sodium channels (Naᵥ) are membrane proteins which open to facilitate the inward flux of sodium ions into excitable cells. In response to stimuli, Naᵥ channels transition from the resting, closed state to an open, conductive state, before rapidly inactivating. Dysregulation of this functional cycle due to mutations causes diseases including epilepsy, pain conditions, and cardiac disorders, making Naᵥ channels a significant pharmacological target. Phosphoinositides are important lipid cofactors for ion channel function. The phosphoinositide PI(4,5)P2 decreases Naᵥ1.4 activity by increasing the difficulty of channel opening, accelerating fast inactivation and slowing recovery from fast inactivation. Using multiscale molecular dynamics simulations, we show that PI(4,5)P2 binds stably to inactivated Naᵥ at a conserved site within the DIV S4–S5 linker, which couples the voltage-sensing domain (VSD) to the pore. As the Naᵥ C-terminal domain is proposed to also bind here during recovery from inactivation, we hypothesize that PI(4,5)P2 prolongs inactivation by competitively binding to this site. In atomistic simulations, PI(4,5)P2 reduces the mobility of both the DIV S4–S5 linker and the DIII–IV linker, responsible for fast inactivation, slowing the conformational changes required for the channel to recover to the resting state. We further show that in a resting state Naᵥ model, phosphoinositides bind to VSD gating charges, which may anchor them and impede VSD activation. Our results provide a mechanism by which phosphoinositides alter the voltage dependence of activation and the rate of recovery from inactivation, an important step for the development of novel therapies to treat Naᵥ-related diseases.

A binding site for phosphoinositides described by multiscale simulations explains their modulation of voltage-gated sodium channels.

Voltage-gated sodium channels (Naᵥ) are membrane proteins which open to facilitate the inward flux of sodium ions into excitable cells. In response to stimuli, Naᵥ channels transition from the resting, closed state to an open, conductive state, before rapidly inactivating. Dysregulation of this functional cycle due to mutations causes diseases including epilepsy, pain conditions, and cardiac disorders, making Naᵥ channels a significant pharmacological target. Phosphoinositides are important lipid cofactors for ion channel function. The phosphoinositide PI(4,5)P2 decreases Naᵥ1.4 activity by increasing the difficulty of channel opening, accelerating fast inactivation and slowing recovery from fast inactivation. Using multiscale molecular dynamics simulations, we show that PI(4,5)P2 binds stably to inactivated Naᵥ at a conserved site within the DIV S4-S5 linker, which couples the voltage-sensing domain (VSD) to the pore. As the Naᵥ C-terminal domain is proposed to also bind here during recovery from inactivation, we hypothesize that PI(4,5)P2 prolongs inactivation by competitively binding to this site. In atomistic simulations, PI(4,5)P2 reduces the mobility of both the DIV S4-S5 linker and the DIII-IV linker, responsible for fast inactivation, slowing the conformational changes required for the channel to recover to the resting state. We further show that in a resting state Naᵥ model, phosphoinositides bind to VSD gating charges, which may anchor them and impede VSD activation. Our results provide a mechanism by which phosphoinositides alter the voltage dependence of activation and the rate of recovery from inactivation, an important step for the development of novel therapies to treat Naᵥ-related diseases.

Trp207 regulation of voltage-dependent activation of human Hv1 proton channel

In voltage-gated Na+ and K+ channels, the hydrophobicity of non-charged residues in the S4 helix has been shown to regulate the S4 movement underlying the process of voltage-sensing domain (VSD) activation. In voltage-gated proton channel Hv1, there is a bulky non-charged tryptophan residue located at the S4 transmembrane segment. This tryptophan remains entirely conserved across all Hv1 members but is not seen in other voltage-gated ion channels, indicating that the tryptophan contributes different roles in VSD activation. The conserved tryptophan of human voltage-gated proton channel Hv1 is Trp207 (W207). Here we showed that W207 modifies human Hv1 voltage-dependent activation, and small residues replacement at position 207 strongly perturbs Hv1 channel opening and closing, the size of the side chain instead of the hydrophobic group of W207 regulates the transition between closed and open states of the channel. We conclude that the large side chain of tryptophan controls the energy barrier during the Hv1 voltage-sensing domain transition.

Effects of electric-field reshaping on voltage sensing in voltage-gated sodium channels.

Voltage-gated sodium channels (Navs) are responsible for the depolarizing phase of the action potential with essential roles in fast electrical signalling. Membrane depolarization triggers opening of Nav channels via activation of their voltage-sensing domains (VSD). The electric field across the membrane acts on a series of positively charged residues and induces their transmembrane relocation, which in turn triggers conformational changes that eventually lead to the opening of the sodium-conducting pore. Voltage dependence of the activation process can be described in terms of the gating charge that links energetics of VSD activation to the transmembrane voltage. Notably, the gating charge is defined by coupling of charged residues to the external electric field, whose shape is therefore crucial for Nav activation. Here, we employed molecular dynamics simulations of cardiac Nav1.5 and bacterial NavAb to gain atomic-level insights into the voltage-sensing mechanisms of Navs. Using our recently developed tool g_elpot to quantify VSD electrostatics with high spatial resolution, we found that, in contrast to earlier low-resolution studies, the electric field within VSDs of Nav channels has a complex isoform- and domain-specific shape, which prominently depends on the activation state of a VSD. Due to this field reshaping, not only translocated basic but also relatively static acidic residues contribute significantly to the gating charge. In the case of NavAb, we found that the transition between the resolved activated- and resting-state structures results in the gating charge of 8e, which is noticeably lower than experimental values of 12-16e. Our analysis of VSD electrostatics thus indicates that the resting-state structure represents an intermediate state of channel activation. In conclusion, our results provide an atomic-level description of the gating charge in Nav channels, and reveal the importance of electric-field reshaping for the energetics of voltage gating.

Molecular Pathology of Sodium Channel Beta-Subunit Variants

The voltage-gated Na+ channel regulates the initiation and propagation of the action potential in excitable cells. The major cardiac isoform NaV1.5, encoded by SCN5A, comprises a monomer with four homologous repeats (I-IV) that each contain a voltage sensing domain (VSD) and pore domain. In native myocytes, NaV1.5 forms a macromolecular complex with NaVβ subunits and other regulatory proteins within the myocyte membrane to maintain normal cardiac function. Disturbance of the NaV complex may manifest as deadly cardiac arrhythmias. Although SCN5A has long been identified as a gene associated with familial atrial fibrillation (AF) and Brugada Syndrome (BrS), other genetic contributors remain poorly understood. Emerging evidence suggests that mutations in the non-covalently interacting NaVβ1 and NaVβ3 are linked to both AF and BrS. Here, we investigated the molecular pathologies of 8 variants in NaVβ1 and NaVβ3. Our results reveal that NaVβ1 and NaVβ3 variants contribute to AF and BrS disease phenotypes by modulating both NaV1.5 expression and gating properties. Most AF-linked variants in the NaVβ1 subunit do not alter the gating kinetics of the sodium channel, but rather modify the channel expression. In contrast, AF-related NaVβ3 variants directly affect channel gating, altering voltage-dependent activation and the time course of recovery from inactivation via the modulation of VSD activation.

Two-stage “Hand-and-Elbow” Gating Mechanism of a KV Channel

During voltage-dependent activation, the voltage-sensing domain (VSD) of voltage-gated potassium (KV) channels activates in a stepwise manner from the resting state to the intermediate state and activated state, which then trigger pore opening via electro-mechanical (E-M) coupling. However, the spatial and temporal details underlying E-M coupling remain elusive. Leveraging the unique two open states of the KV7.1 channel that are associated with the VSD adopting the intermediate and activated conformations, here we report a two-stage E-M coupling mechanism in KV7.1 as triggered by the two-steps VSD activation. When the S4 segment transitions to the intermediate state, the hand-like C-terminus of the VSD-pore linker (S4-S5L) interacts with the pore in the same subunit. When S4 then proceeds on to the activated state, the elbow-like hinge between S4 and S4-S5L engages with the pore to activate conductance. This two-stage “hand-and-elbow” gating mechanism elucidates distinct tissue-specific modulations, pharmacology, and disease pathogenesis of KV7.1, and likely applies to numerous KV channels.

Calcium-driven regulation of voltage-sensing domains in BK channels.

Allosteric interactions between the voltage-sensing domain (VSD), the Ca2+-binding sites, and the pore domain govern the mammalian Ca2+- and voltage-activated K+ (BK) channel opening. However, the functional relevance of the crosstalk between the Ca2+- and voltage-sensing mechanisms on BK channel gating is still debated. We examined the energetic interaction between Ca2+ binding and VSD activation by investigating the effects of internal Ca2+ on BK channel gating currents. Our results indicate that Ca2+ sensor occupancy has a strong impact on VSD activation through a coordinated interaction mechanism in which Ca2+ binding to a single α-subunit affects all VSDs equally. Moreover, the two distinct high-affinity Ca2+-binding sites contained in the C-terminus domains, RCK1 and RCK2, contribute equally to decrease the free energy necessary to activate the VSD. We conclude that voltage-dependent gating and pore opening in BK channels is modulated to a great extent by the interaction between Ca2+ sensors and VSDs.

ML277 specifically enhances the fully activated open state of KCNQ1 by modulating VSD-pore coupling.

Upon membrane depolarization, the KCNQ1 potassium channel opens at the intermediate (IO) and activated (AO) states of the stepwise voltage-sensing domain (VSD) activation. In the heart, KCNQ1 associates with KCNE1 subunits to form IKs channels that regulate heart rhythm. KCNE1 suppresses the IO state so that the IKs channel opens only to the AO state. Here, we tested modulations of human KCNQ1 channels by an activator ML277 in Xenopus oocytes. It exclusively changes the pore opening properties of the AO state without altering the IO state, but does not affect VSD activation. These observations support a distinctive mechanism responsible for the VSD-pore coupling at the AO state that is sensitive to ML277 modulation. ML277 provides insights and a tool to investigate the gating mechanism of KCNQ1 channels, and our study reveals a new strategy for treating long QT syndrome by specifically enhancing the AO state of native IKs currents.

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.

Mechanisms Responsible for ω-Pore Currents in Cav Calcium Channel Voltage-Sensing Domains

Mutations of positively charged amino acids in the S4 transmembrane segment of a voltage-gated ion channel form ion-conducting pathways through the voltage-sensing domain, named ω-current. Here, we used structure modeling and MD simulations to predict pathogenic ω-currents in CaV1.1 and CaV1.3 Ca2+ channels bearing several S4 charge mutations. Our modeling predicts that mutations of CaV1.1-R1 (R528H/G, R897S) or CaV1.1-R2 (R900S, R1239H) linked to hypokalemic periodic paralysis type 1 and of CaV1.3-R3 (R990H) identified in aldosterone-producing adenomas conducts ω-currents in resting state, but not during voltage-sensing domain activation. The mechanism responsible for the ω-current and its amplitude depend on the number of charges in S4, the position of the mutated S4 charge and countercharges, and the nature of the replacing amino acid. Functional characterization validates the modeling prediction showing that CaV1.3-R990H channels conduct ω-currents at hyperpolarizing potentials, but not upon membrane depolarization compared with wild-type channels.

Distance-Resolving Voltage Clamp Fluorometry (drVCF) Quantifies Intramolecular Transitions in the Human BK and CI-VSP Voltage Sensors Under Physiologically-Relevant Conditions

The BK channel voltage-sensing domain (VSD) exhibits important differences from the canonical VSD model: an additional helix (S0), a decentralized distribution of voltage-sensing charges throughout S2-S3-S4 and cooperative S2/S4 activation transitions. To understand the voltage-evoked conformational rearrangements of this unique voltage sensor, and address the unmet need of quantifying short-range intramolecular distances in conducting channels under physiological conditions, we have been developing a new theoretical and experimental framework: distance-resolving Voltage Clamp Fluorometry (drVCF).drVCF exploits state-dependent collisional quenching of site-specifically conjugated tetramethylrhodamine fluorophores of different length by tryptophan. Firstly, voltage-evoked, Trp-dependent fluorescence deflections of each label are acquired from the VSD of conducting human BK channels expressed in Xenopus oocytes using VCF-enabled cut-open oocyte voltage clamp. The labeling site/tryptophan distance in the resting and active states is determined by fitting VCF data to quenching probability density over distance, generated by fluorophore molecular dynamics simulations. Simultaneous fitting of data from multiple fluorophores results in well-constrained solutions. Confidence intervals are established using bootstrap resampling.At rest, S0, S1 and S2 are approximately equidistant from S4: S0-S4 mean=12.5A; 95% CI [12.4-12.8A]; S1-S4=11.9A [10.4-12.9A]; S2-S4=11.5A [8.8-12.3A]. Upon VSD activation, S4 diverges from S0 (22.0A [21.4-23.1A]) and S1 (18.4A [17.7-20.0A]), while the S2-S4 distance exceeds 24A. Using this information, we constructed a structural model for the BK voltage sensor in the resting and active states. Applying drVCF in Ci-VSP, we resolved that positions 137 (S1) and 212 (S4) are 19-24A apart in the active state, as in the atomic structure (22A: Li et al., 2014; PDB#4G7V). Thus, the new drVCF approach complements FRET-based techniques for distance measurements of short-range transitions under physiologically-relevant conditions.

Probing Mechanisms through which Drugs Affect Voltage-Sensitive Gating

Voltage-gated ion channels, which serve as the molecular basis for electrical signaling in excitable cells, represent a valuable target for drug therapy. However, there is a void in the mechanistic understanding of drug action on voltage-dependent gating. In our recent work on PIP2 regulation of Kv7.1, we developed a methodology to isolate the effect of PIP2 on three fundamental gating processes: voltage-sensing domain (VSD) activation, pore-gate domain (PGD) opening, and PGD-VSD coupling. Using Voltage Clamp Fluorometry (VCF) to track VSD movement simultaneously with ionic current to monitor PGD opening, we were able to gauge PIP2 effects on PGD opening and VSD activation. We detected coupling by performing VCF on locked-open channel mutants, allowing us to directly observe the impact of pore-opening on VSD activation through the VSD-PGD coupling. Here we demonstrate the ability of our methodology to provide mechanistic insights into drug effects using the potent Kv7.1 channel activator ML277 (Mattmann 2012). As reported previously, 10 μM ML277 caused a maximal channel potentiation of around 4.5x initial current. We observed channel potentiation at 40 mV, at which point VSD activation is complete, indicating that ML277 acts on processes other than VSD activation. Using VCF, we see that ML277 mildly right-shifts VSD activation in WT channels. This result contradicts pore-opener behavior, which would have left-shifted the fluorescence-voltage (FV) relationship. In contrast we see a dramatic left-shift in the FV relationship of the lock-open mutant, leading to the conclusion that ML277 strengthens the coupling process. Consistently, we saw a higher apparent affinity for ML277 in Kv380 mutants, which have been shown to exhibit increased PIP2 affinity. This study shows the utility of our methodology to analyze drug effects on voltage-sensitive gating.

Ph Sensitivity of Voltage Sensing Domain Relaxation

Voltage sensing domain (VSD) relaxation is a process involving a voltage-independent transition from the activated state into the relaxed state. This process has been shown to shift the voltage dependence for sensing charge movement (sensing current) in the voltage sensitive phosphatase (VSP) known as Ci-VSP and to slow down the deactivation of potassium currents in Shaker and Kv1.2. Given the effect of VSD relaxation on the dynamics of these voltage sensitive proteins, it is thought that the relaxed and the active states are comprised of different set of conformations. Thus, it is possible that these states are sensitive to different physiological parameters. Here, it is shown that VSD activation and relaxation in Ci-VSP display different sensitivity to external pH (pHEXT). Sensing currents of Ci-VSP recorded during activation slowed down as pHEXT was decreased. Noteworthy, the kinetic of deactivating sensing currents was less affected, suggesting that the initial activation of the VSD involved a pHEXT-sensitive, rate-limiting step. In spite of these observations, the voltage dependence of activating sensing currents was almost unaltered by pHEXT (6.5 to 9.0). In contrast, voltage dependence was strongly modulated by pHEXT after VSD relaxation. In fact, the shift in voltage dependence for sensing current was 30 mV larger at pHEXT 6.5 with respect to 9.0. These observations indicated that the equilibrium between the resting and active states was pHEXT insensitive. Conversely, the equilibrium between the relaxed and the relaxed-resting states was unambiguously pHEXT sensitive. Furthermore, given that decreasing pHEXT slowed sensing currents, this observation suggests that the energy barrier between the resting and active states, rather than the difference in energy between these states, was modulated by pHEXT. In contrast, for the relaxed VSD, pHEXT strongly modulated the difference in energy between the relaxed and the relaxed-resting states.

Multi-Dimensional Free Energy Landscape of Voltage Sensor Domain Activation

Many modulators, such as toxins, anesthetics or drugs, act on voltage-gated channels by altering the activation and/or deactivation mechanism of their voltage-sensing domains (VSD). So far, we have proposed a static model of the Kv1.2 VSD activation using brute force and modified molecular dynamics simulations and that agrees with a large body of experimental data. This model involves 5 states: α (activated), β, γ, δ (three intermediate) and e (resting) [Delemotte et al. 2011, Proc. Natl. Acad. Sci. USA, 108:6109-6114], and has enabled to gain access to the contribution of transmembrane voltage to the free energy of activation via calculation of the corresponding gating charge. Crucial details, however, are still missing, among which an estimation of the thermodynamic stability of these states or of the minimum energy transition pathway linking them. In order to complete our understanding of VSD function, we produce the multi-dimensional free energy landscape (FES) of the four transitions linking the Kv1.2 VSD conformations, enabling to follow for the first time the pathway of activation of a VSD. We then investigate how the free energy landscape of VSD activation is modified by a change in the lipid environment and by the mutation of key residues.

The α2δ Subunit Facilitates CaV1.2 Channel Activation by Remodeling Its Four Voltage Sensor Domains

Voltage-gated L-type calcium channels (CaV1.2) are multimeric proteins composed of the pore-forming α1C subunit and at least two ancillary subunits, β and α2δ, in a 1:1:1 stoichiometry. The α2δ subunit is a large extracellular protein with a membrane-anchoring segment. This auxiliary subunit not only promotes channel trafficking to the plasma membrane, but also facilitates channel activation, shifting the voltage dependence of channel opening toward more negative potentials. The underlying mechanism by which the α2δ subunit facilitates CaV1.2 activation is unknown. Using the voltage clamp fluorometry technique, we fluorescently tracked the molecular rearrangements of the four individual CaV1.2 voltage-sensing domains (VSDs) in the presence or in the absence of the α2δ subunit. We found that this subunit facilitated the activation of all VSDs, as revealed by a more hyperpolarized voltage dependence of VSD activation (FV), although to a different extent: the FV shift was ∼-33 mV for VSD I, ∼-19 mV for VSD II, ∼-16 mV for VSD III and ∼-96 mV for VSD IV, resulting in ∼0.3 probability of VSD IV activation at resting potential (−80 mV). The α2δ subunit also shifted the voltage dependence of the conductance (GV) towards more hyperpolarized membrane potentials (∼-70 mV). That is, the presence of α2δ subunits profoundly remodels the voltage sensor domains of CaV1.2 channels, facilitating voltage dependent activation. This facilitation emerges from distinct functional interactions between the α2δ subunit and individual CaV1.2 VSDs, consistent with α2δ asymmetric assembly with the α1C subunit. The energetic contribution of each VSD to pore opening was quantified using linkage analysis combined with an allosteric model that consists of five gating particle (one pore and four VSDs). Funded by: NIH, AHA, FONDECYT, ACT.

PIP2 is Required to Couple the Voltage-Sensing Domain to the Pore for the Activation of KCNQ1 by Voltage

Voltage-gated ion channels are vital for the generation of action potentials that orchestrate various physiological processes. Phosphatidylinositol 4,5-bisphophate (PIP2) is a membrane lipid that modulates several voltage-gated channels (TRP, HCN, CaV, Kv, KCNQ). KCNQ channels are unique in that they cannot be activated by voltage in the absence of PIP2 and the loss of KCNQ channel activity due to PIP2 depletion is known to be physiologically relevant. Although residues governing PIP2 sensitivity have been identified for several KCNQ channels, it is unclear where PIP2 binds and what it does to channel gating. Voltage-dependent gating involves coupled conformational changes in the voltage-sensing domain (VSD) and the pore domain (PD) providing three possible mechanisms of PIP2 action: VSD activation, PD opening, or coupling between VSD activation and PD opening. We combine voltage-clamp fluorometry wih expression of a voltage-sensitive lipid-phosphatase in order to measure VSD activation and PD opening simultaneously while manipulating PIP2 abundance. We find that PIP2 is not necessary for VSD activation; however, PIP2 is required for VSD-PD coupling. Deriving a simple mathematical model, we find that PIP2 regulation of coupling is sufficient to recapitulate the experimental results without any direct effects on PD opening. These findings suggest PIP2 may bind at the VSD-PD interface that, in KCNQ1, is densely populated with basic residues near the intracellular face of the membrane. By mutating these residues and measuring the effects on channel function, PIP2 sensitivity and VSD-PD coupling, we propose a PIP2 binding site. In our resulting model, the negatively charged PIP2 headgroup resolves the electrostatic repulsion between basic residues on the VSD and the PD, thus holding the two domains together and allowing the transfer of conformational energy between them.

Voltage Gated Cation Channels Activation: Towards an Ab-Initio Kinetic Model

Many modulators, such as toxins, anesthetics or drugs, act on voltage-gated cation channels (VGCCs) by altering the kinetics of activation and/or deactivation of their voltage-sensing domains (VSD). So far, we have proposed a “static” model of the Kv1.2 VSD activation using brute force and modified molecular dynamics simulations and that agrees with a large body of experimental data. This model involves 5 states: α (activated), β, γ, δ (three intermediate) and e (resting) [Delemotte et al. 2011, Proc. Natl. Acad. Sci. USA, 108:6109-6114], and has enabled to gain access to the contribution of transmembrane voltage to the free energy of activation via calculation of the corresponding gating charge. Crucial details, however, are still missing, among which an estimation of the thermodynamic and kinetic stability of these states or of the minimum energy transition pathway linking them.In order to complete our understanding of VSD function, we produce the free energy landscape (FES) of the four transitions linking the Kv1.2 VSD conformations and estimate therefrom the corresponding the rate (kinetic) constants. This enables, not only to follow for the first time the pathway of activation of a VGCC VSD, but also to produce a complete ab-initio kinetic model of its activation based uniquely on parameters derived from an in-silico investigation. The G/V and Q/V curves (ionic current and gating current/voltage, respectively) characteristic of the function of wild type channels derived therefrom are then compared to electrophysiology recordings. The study is then extendable to investigate the modulation of VSD function by drugs, toxins, mutations or else lipid interaction.

The Contribution of RCK Domains to Human BK Channel Allosteric Activation

Large conductance voltage- and Ca(2+)-activated K(+) (BK) channels are potent regulators of cellular processes including neuronal firing, synaptic transmission, cochlear hair cell tuning, insulin release, and smooth muscle tone. Their unique activation pathway relies on structurally distinct regulatory domains including one transmembrane voltage-sensing domain (VSD) and two intracellular high affinity Ca(2+)-sensing sites per subunit (located in the RCK1 and RCK2 domains). Four pairs of RCK1 and RCK2 domains form a Ca(2+)-sensing apparatus known as the "gating ring." The allosteric interplay between voltage- and Ca(2+)-sensing apparati is a fundamental mechanism of BK channel function. Using voltage-clamp fluorometry and UV photolysis of intracellular caged Ca(2+), we optically resolved VSD activation prompted by Ca(2+) binding to the gating ring. The sudden increase of intracellular Ca(2+) concentration ([Ca(2+)](i)) induced a hyperpolarizing shift in the voltage dependence of both channel opening and VSD activation, reported by a fluorophore labeling position 202, located in the upper side of the S4 transmembrane segment. The neutralization of the Ca(2+) sensor located in the RCK2 domain abolished the effect of [Ca(2+)](i) increase on the VSD rearrangements. On the other hand, the mutation of RCK1 residues involved in Ca(2+) sensing did not prevent the effect of Ca(2+) release on the VSD, revealing a functionally distinct interaction between RCK1 and RCK2 and the VSD. A statistical-mechanical model quantifies the complex thermodynamics interplay between Ca(2+) association in two distinct sites, voltage sensor activation, and BK channel opening.

The Role of PIP2 in the Voltage-Dependent Activation of Kv7.1

Phosphatidylinositol 4,5-bisphosphate (PIP2) is an important regulator of ion channel activity. While PIP2 modulation of voltage-independent Kir channels has been extensively studied, the mechanism of how PIP2 potentiates the activity of voltage-dependent ion channels remains uknown. In this work, we study PIP2 activation of a voltage-dependent potassium channel (Kv7.1). Kv7.1 channels have a canonical Kv structure with a central pore-gate domain (PGD) surrounded by four peripheral voltage-sensing domains (VSDs). Kv channel activation is thought to involve two general steps. First, membrane depolarization is sensed by gating charges in the VSDs resulting in conformational changes within the VSDs from the resting state to the activated state. After all four VSDs have been activated, the second general step in Kv activation involves a concerted motion during which the PGD opens to allow ion permeation. In this study we ask if PIP2 regulates activation of Kv7.1 by potentiating the early VSD conformational changes, the concerted opening of the PGD, or both. Using the voltage-clamp fluorometry (VCF) technique to assay local conformation changes in the VSDs and the PGD simultaneously, we are able to show that although depletion of PIP2 eliminates the ionic current, the early conformational changes in the VSD do not require PIP2. This result indicates that the later conformational changes that couple VSD activation to the opening of the PGD are eliminated by PIP2 depletion. We continue this work by dissecting the molecular details of why PIP2 is required for opening of the PGD in response to VSD activation. These results may provide insights on the common principle of how Kv channels are modulated by PIP2.

Regulation of Voltage-Activated K+ Channel Gating by Transmembrane β Subunits

Voltage-activated K+ (KV) channels are important for shaping action potentials and maintaining resting membrane potential in excitable cells. KV channels contain a central pore-gate domain (PGD) surrounded by four voltage-sensing domains (VSDs). The VSDs will change conformation in response to alterations of the membrane potential thereby inducing the opening of the PGD. Many KV channels are heteromeric protein complexes containing auxiliary β subunits. These β subunits modulate channel expression and activity to increase functional diversity and render tissue specific phenotypes. This review focuses on the KV β subunits that contain transmembrane (TM) segments including the KCNE family and the β subunits of large conductance, Ca2+- and voltage-activated K+ (BK) channels. These TM β subunits affect the voltage-dependent activation of KV α subunits. Experimental and computational studies have described the structural location of these β subunits in the channel complexes and the biophysical effects on VSD activation, PGD opening, and VSD–PGD coupling. These results reveal some common characteristics and mechanistic insights into KV channel modulation by TM β subunits.