Positive Allosteric Modulation of Kv Channels by Sevoflurane: Insights into the Structural Basis of Inhaled Anesthetic Action

Inhalational general anesthesia results from the poorly understood interactions of haloethers with multiple protein targets, which prominently includes ion channels in the nervous system. Previously, we reported that the commonly used inhaled anesthetic sevoflurane potentiates the activity of voltage-gated K⁺ (Kv) channels, specifically, several mammalian Kv1 channels and the Drosophila K-Shaw2 channel. Also, previous work suggested that the S4-S5 linker of K-Shaw2 plays a role in the inhibition of this Kv channel by n-alcohols and inhaled anesthetics. Here, we hypothesized that the S4-S5 linker is also a determinant of the potentiation of Kv1.2 and K-Shaw2 by sevoflurane. Following functional expression of these Kv channels in Xenopus oocytes, we found that converse mutations in Kv1.2 (G329T) and K-Shaw2 (T330G) dramatically enhance and inhibit the potentiation of the corresponding conductances by sevoflurane, respectively. Additionally, Kv1.2-G329T impairs voltage-dependent gating, which suggests that Kv1.2 modulation by sevoflurane is tied to gating in a state-dependent manner. Toward creating a minimal Kv1.2 structural model displaying the putative sevoflurane binding sites, we also found that the positive modulations of Kv1.2 and Kv1.2-G329T by sevoflurane and other general anesthetics are T1-independent. In contrast, the positive sevoflurane modulation of K-Shaw2 is T1-dependent. In silico docking and molecular dynamics-based free-energy calculations suggest that sevoflurane occupies distinct sites near the S4-S5 linker, the pore domain and around the external selectivity filter. We conclude that the positive allosteric modulation of the Kv channels by sevoflurane involves separable processes and multiple sites within regions intimately involved in channel gating.     

A hot spot for the interaction of gating modifier toxins with voltage-dependent ion channels

The gating modifier toxins are a large family of protein toxins that modify either activation or inactivation of voltage-gated ion channels. omega-Aga-IVA is a gating modifier toxin from spider venom that inhibits voltage-gated Ca(2+) channels by shifting activation to more depolarized voltages. We identified two Glu residues near the COOH-terminal edge of S3 in the alpha(1A) Ca(2+) channel (one in repeat I and the other in repeat IV) that align with Glu residues previously implicated in forming the binding sites for gating modifier toxins on K(+) and Na(+) channels. We found that mutation of the Glu residue in repeat I of the Ca(2+) channel had no significant effect on inhibition by omega-Aga-IVA, whereas the equivalent mutation of the Glu in repeat IV disrupted inhibition by the toxin. These results suggest that the COOH-terminal end of S3 within repeat IV contributes to forming a receptor for omega-Aga-IVA. The strong predictive value of previous mapping studies for K(+) and Na(+) channel toxins argues for a conserved binding motif for gating modifier toxins within the voltage-sensing domains of voltage-gated ion channels.     

Modeling of benzocaine analog interactions with the D4S6 segment of NaV4.1 voltage-gated sodium channels

Local anesthetics (LAs) are compounds that inhibit the propagation of action potentials in excitable tissues by blocking voltage-gated Na+ channels. Mutagenesis studies have demonstrated that several amino acid residues are important sites of LA interaction with the channel, but these studies provide little information regarding the molecular forces that govern drug-binding interactions, including the binding orientation of drugs. We used computational methods to construct a simple model of benzocaine analog binding with the D4S6 segment of rat skeletal muscle (NaV4.1) sodium channels. The model revealed that four hydrophobic residues form a binding cavity for neutral LAs, and docking studies indicated that increasing hydrophobicity among the benzocaine analogs allowed a better fit within the binding cavity. The similarities between our simple model and published experimental data suggested that modeling of LA interactions with sodium channels, along with experimental approaches, could further enhance our understanding of LA interactions with sodium channels.     

State-dependent inhibition of sodium channels by local anesthetics: A 40-year evolution

Knowledge about the mechanism of impulse blockade by local anesthetics has evolved over the past four decades, from the realization that Na⁺ channels were inhibited to affect the impulse blockade to an identification of the amino acid residues within the Na⁺ channel that bind the local anesthetic molecule. Within this period appreciation has grown of the state-dependent nature of channel inhibition, with rapid binding and unbinding at relatively high affinity to the open state, and weaker binding to the closed resting state. Slow binding of high affinity for the inactivated state accounts for the salutary therapeutic as well as the toxic actions of diverse class I anti-arrhythmic agents, but may have little importance for impulse blockade, which requires concentrations high enough to block the resting state. At the molecular level, residues on the S6 transmembrane segments in three of the homologous domains of the channel appear to contribute to the binding of local anesthetics, with some contribution also from parts of the selectivity filter. Binding to the inactivated state, and perhaps the open state, involves some residues that are not identical to those that bind these drugs in the resting state, suggesting spatial flexibility in the “binding site”. Questions remaining include the mechanism that links local anesthetic binding with the inhibition of gating charge movements, and the molecular nature of the theoretical “hydrophobic pathway” that may be critical for determining the recovery rates from blockade of closed channels, and thus account for both therapeutic and cardiotoxic actions.     

Residues in Na(+) channel D3-S6 segment modulate both batrachotoxin and local anesthetic affinities

Batrachotoxin (BTX) alters the gating of voltage-gated Na(+) channels and causes these channels to open persistently, whereas local anesthetics (LAs) block Na(+) conductance. The BTX and LA receptors have been mapped to several common residues in D1-S6 and D4-S6 segments of the Na(+) channel alpha-subunit. We substituted individual residues with lysine in homologous segment D3-S6 of the rat muscle mu1 Na(+) channel from F1274 to N1281 to determine whether additional residues are involved in BTX and LA binding. Two mutant channels, mu1-S1276K and mu1-L1280K, when expressed in mammalian cells, become completely resistant to 5 microM BTX during repetitive pulses. The activation and/or fast inactivation gating of these mutants is substantially different from that of wild type. These mutants also display approximately 10-20-fold reduction in bupivacaine affinity toward their inactivated state but show only approximately twofold affinity changes toward their resting state. These results demonstrate that residues mu1-S1276 and mu1-L1280 in D3-S6 are critical for both BTX and LA binding interactions. We propose that LAs interact readily with these residues from D3-S6 along with those from D1-S6 and D4-S6 in close proximity when the Na(+) channel is in its inactivated state. Implications of this state-dependent binding model for the S6 alignment are discussed.     

Anesthetic Binding in a Pentameric Ligand-Gated Ion Channel: GLIC

Cys-loop receptors are molecular targets of general anesthetics, but the knowledge of anesthetic binding to these proteins remains limited. Here we investigate anesthetic binding to the bacterial Gloeobacter violaceus pentameric ligand-gated ion channel (GLIC), a structural homolog of cys-loop receptors, using an experimental and computational hybrid approach. Tryptophan fluorescence quenching experiments showed halothane and thiopental binding at three tryptophan-associated sites in the extracellular (EC) domain, transmembrane (TM) domain, and EC-TM interface of GLIC. An additional binding site at the EC-TM interface was predicted by docking analysis and validated by quenching experiments on the N200W GLIC mutant. The binding affinities (K_D) of 2.3 ± 0.1 mM and 0.10 ± 0.01 mM were derived from the fluorescence quenching data of halothane and thiopental, respectively. Docking these anesthetics to the original GLIC crystal structure and the structures relaxed by molecular dynamics simulations revealed intrasubunit sites for most halothane binding and intersubunit sites for thiopental binding. Tryptophans were within reach of both intra- and intersubunit binding sites. Multiple molecular dynamics simulations on GLIC in the presence of halothane at different sites suggested that anesthetic binding at the EC-TM interface disrupted the critical interactions for channel gating, altered motion of the TM23 linker, and destabilized the open-channel conformation that can lead to inhibition of GLIC channel current. The study has not only provided insights into anesthetic binding in GLIC, but also demonstrated a successful fusion of experiments and computations for understanding anesthetic actions in complex proteins.     

Modification of cardiac Na+ channels by batrachotoxin: effects on gating, kinetics, and local anesthetic binding.

The purpose of the present study was to examine the characteristics of Na+ channel modification by batrachotoxin (BTX) in cardiac cells, including changes in channel gating and kinetics as well as susceptibility to block by local anesthetic agents. We used the whole cell configuration of the patch clamp technique to measure Na+ current in guinea pig myocytes. Extracellular Na+ concentration and temperature were lowered (5-10 mM, 17 degrees C) in order to maintain good voltage control. Our results demonstrated that 1) BTX modifies cardiac INa, causing a substantial steady-state (noninactivating) component of INa, 2) modification of cardiac Na+ channels by BTX shifts activation to more negative potentials and reduces both maximal gNa and selectivity for Na+; 3) binding of BTX to its receptor in the cardiac Na+ channel reduces the affinity of local anesthetics for their binding site; and 4) BTX-modified channels show use-dependent block by local anesthetics. The reduced blocking potency of local anesthetics for BTX-modified Na+ channels probably results from an allosteric interaction between BTX and local anesthetics for their respective binding sites in the Na+ channel. Our observations that use-dependent block by local anesthetics persists in BTX-modified Na+ channels suggest that this form of extra block can occur in the virtual absence of the inactivated state. Thus, the development of use-dependent block appears to rely primarily on local anesthetic binding to activated Na+ channels under these conditions.     

New insights into the therapeutic inhibition of voltage-gated sodium channels

Antiarrhythmics, anticonvulsants and local anesthetics inhibit voltage-gated sodium channels and reduce membrane excitability in neurons and muscle, making them useful in the management of cardiac arrhythmias, epilepsy and pain. These compounds, which are often termed singly in the literature as 'local anesthetics', have at least two inhibitory states: a resting inhibition that develops with intermittent stimulation and a higher affinity inhibition that arises upon repeated depolarization and likely involves the inactivated state of the channel. Although elucidating their mechanism of inhibition has been an active area of research for decades, many questions remain unanswered. Do these two inhibitory states share a common, but guarded or modulated receptor? Or do they represent different protonated states of the drugs, many of which have pKa's close to physiological pH, thereby yielding a significant population of both charged and uncharged compound inside cells. Some mechanistic clues can be found by mutating conserved phenylalanine and tyrosine residues of the 'local anesthetic receptor' in the channel's inner vestibule. Mutations of these aromatic residues universally disrupt the mechanism of drug inhibition in numerous channel isoforms. For instance, non aromatic substitutions of Phe1579 (Na(V) numbering) in the pore lining S6 segment of domain four (DIVS6) can abolish inactivated state inhibition.(1,2) The strict conservation of Phe1579 and other DIVS6 aromatic residues in all nine sodium channel isoforms led us to further dissect the role of this and other aromatic residues on local anesthetic inhibition. We recently employed subtly modified phenylalanine derivatives to better understand the role of these aromatics in the binding of local anesthetics and found a significant electrostatic interaction at one site, Phe1579, contributes to channel inhibition.(3) What follows is a self guided tour of our motivation and experimental findings.     

Resin-acid derivatives bind to multiple sites on the voltage-sensor domain of the Shaker potassium channel

Voltage-gated potassium (K_V) channels can be opened by negatively charged resin acids and their derivatives. These resin acids have been proposed to attract the positively charged voltage-sensor helix (S4) toward the extracellular side of the membrane by binding to a pocket located between the lipid-facing extracellular ends of the transmembrane segments S3 and S4. By contrast to this proposed mechanism, neutralization of the top gating charge of the Shaker K_V channel increased resin-acid–induced opening, suggesting other mechanisms and sites of action. Here, we explore the binding of two resin-acid derivatives, Wu50 and Wu161, to the activated/open state of the Shaker K_V channel by a combination of in silico docking, molecular dynamics simulations, and electrophysiology of mutated channels. We identified three potential resin-acid–binding sites around S4: (1) the S3/S4 site previously suggested, in which positively charged residues introduced at the top of S4 are critical to keep the compound bound, (2) a site in the cleft between S4 and the pore domain (S4/pore site), in which a tryptophan at the top of S6 and the top gating charge of S4 keeps the compound bound, and (3) a site located on the extracellular side of the voltage-sensor domain, in a cleft formed by S1–S4 (the top-VSD site). The multiple binding sites around S4 and the anticipated helical-screw motion of the helix during activation make the effect of resin-acid derivatives on channel function intricate. The propensity of a specific resin acid to activate and open a voltage-gated channel likely depends on its exact binding dynamics and the types of interactions it can form with the protein in a state-specific manner.     

Interactions of Local Anesthetics with Voltage-gated Na<Superscript>+</Superscript> Channels

Voltage-gated Na⁺ channels are dynamic transmembrane proteins responsible for the rising phase of the action potential in excitable membranes. Local anesthetics (LAs) and structurally related antiarrhythmic and anticonvulsant compounds target specific sites in voltage-gated Na⁺ channels to block Na⁺ currents, thus reducing excitability in neuronal, cardiac, or central nervous tissue. A high-affinity LA block is produced by binding to open and inactivated states of Na⁺ channels rather than to resting states and suggests a binding site that converts from a low- to a high-affinity conformation during gating. Recent findings using site-directed mutagenesis suggest that multiple S6 segments together form an LA binding site within the Na⁺ channel. While the selectivity filter may form the more extracellular-located part of this binding site, the role of the fast inactivation gate in LA binding has not yet been resolved. The receptor of the neurotoxin batrachotoxin (BTX) is adjacent to or even overlaps with the LA binding site. The close proximity of the LA and BTX binding sites to residues critical for inactivation, together with gating transitions through S6 segments, might explain the strong impact of LAs and BTX on inactivation of voltage-gated Na⁺ channels and might help elucidate the mechanisms underlying voltage- and frequency-dependent LA block.     

Isoflurane alters the structure and dynamics of GLIC

Pentameric ligand-gated ion channels are targets of general anesthetics. Although the search for discrete anesthetic binding sites has achieved some degree of success, little is known regarding how anesthetics work after the events of binding. Using the crystal structures of the bacterial Gloeobacter violaceus pentameric ligand-gated ion channel (GLIC), which is sensitive to a variety of general anesthetics, we performed multiple molecular dynamics simulations in the presence and absence of the general anesthetic isoflurane. Isoflurane bound to several locations within GLIC, including the transmembrane pocket identified crystallographically, the extracellular (EC) domain, and the interface of the EC and transmembrane domains. Isoflurane also entered the channel after the pore was dehydrated in one of the simulations. Isoflurane disrupted the quaternary structure of GLIC, as evidenced in a striking association between the binding and breakage of intersubunit salt bridges in the EC domain. The pore-lining helix experienced lateral and inward radial tilting motion that contributed to the channel closure. Isoflurane binding introduced strong anticorrelated motions between different subunits of GLIC. The demonstrated structural and dynamical modulations by isoflurane aid in the understanding of the underlying mechanism of anesthetic inhibition of GLIC and possibly other homologous pentameric ligand-gated ion channels.     

Role of charged residues in the S1-S4 voltage sensor of BK channels

The activation of large conductance Ca(2+)-activated (BK) potassium channels is weakly voltage dependent compared to Shaker and other voltage-gated K(+) (K(V)) channels. Yet BK and K(V) channels share many conserved charged residues in transmembrane segments S1-S4. We mutated these residues individually in mSlo1 BK channels to determine their role in voltage gating, and characterized the voltage dependence of steady-state activation (P(o)) and I(K) kinetics (tau(I(K))) over an extended voltage range in 0-50 microM [Ca(2+)](i). mSlo1 contains several positively charged arginines in S4, but only one (R213) together with residues in S2 (D153, R167) and S3 (D186) are potentially voltage sensing based on the ability of charge-altering mutations to reduce the maximal voltage dependence of P(O). The voltage dependence of P(O) and tau(I(K)) at extreme negative potentials was also reduced, implying that the closed-open conformational change and voltage sensor activation share a common source of gating charge. Although the position of charged residues in the BK and K(V) channel sequence appears conserved, the distribution of voltage-sensing residues is not. Thus the weak voltage dependence of BK channel activation does not merely reflect a lack of charge but likely differences with respect to K(V) channels in the position and movement of charged residues within the electric field. Although mutation of most sites in S1-S4 did not reduce gating charge, they often altered the equilibrium constant for voltage sensor activation. In particular, neutralization of R207 or R210 in S4 stabilizes the activated state by 3-7 kcal mol(-1), indicating a strong contribution of non-voltage-sensing residues to channel function, consistent with their participation in state-dependent salt bridge interactions. Mutations in S4 and S3 (R210E, D186A, and E180A) also unexpectedly weakened the allosteric coupling of voltage sensor activation to channel opening. The implications of our findings for BK channel voltage gating and general mechanisms of voltage sensor activation are discussed.     

Exploring Volatile General Anesthetic Binding to a Closed Membrane-Bound Bacterial Voltage-Gated Sodium Channel via Computation

Despite the clinical ubiquity of anesthesia, the molecular basis of anesthetic action is poorly understood. Amongst the many molecular targets proposed to contribute to anesthetic effects, the voltage gated sodium channels (VGSCs) should also be considered relevant, as they have been shown to be sensitive to all general anesthetics tested thus far. However, binding sites for VGSCs have not been identified. Moreover, the mechanism of inhibition is still largely unknown. The recently reported atomic structures of several members of the bacterial VGSC family offer the opportunity to shed light on the mechanism of action of anesthetics on these important ion channels. To this end, we have performed a molecular dynamics “flooding” simulation on a membrane-bound structural model of the archetypal bacterial VGSC, NaChBac in a closed pore conformation. This computation allowed us to identify binding sites and access pathways for the commonly used volatile general anesthetic, isoflurane. Three sites have been characterized with binding affinities in a physiologically relevant range. Interestingly, one of the most favorable sites is in the pore of the channel, suggesting that the binding sites of local and general anesthetics may overlap. Surprisingly, even though the activation gate of the channel is closed, and therefore the pore and the aqueous compartment at the intracellular side are disconnected, we observe binding of isoflurane in the central cavity. Several sampled association and dissociation events in the central cavity provide consistent support to the hypothesis that the “fenestrations” present in the membrane-embedded region of the channel act as the long-hypothesized hydrophobic drug access pathway.     

Differences Between Ion Binding to eag and HERG voltage sensors Contribute to Differential Regulation of Activation and Deactivation Gating

HERG (KCNH2) and ether-à-go-go (eag) (KCNH1) are members of the same subfamily of voltage-gated K+ channels. In eag, voltage-dependent activation is significantly slowed by extracellular divalent cations. To exert this effect, ions bind to a site located between transmembrane segments S2 and S3 in the voltage sensor domain where they interact with acidic residues that are conserved only among members of the eag subfamily. In HERG channels, extracellular divalent ions significantly accelerate deactivation. To investigate the ion-binding site in HERG, acidic residues in S2 and S3 were neutralized singly or in pairs to alanine, and the functional effects of extracellular Mg2+ were characterized in Xenopus oocytes. To modulate deactivation kinetics in HERG, divalent cations interact with eag subfamily-specific acidic residues (D460 and D509) and also with an acidic residue in S2 (D456) that is widely conserved in the voltage-gated channel superfamily. In contrast, the analogous widely-conserved residue does not contribute to the ion-binding site that modulates activation kinetics in eag. We propose that structural differences between the ion-binding sites in the eag and HERG voltage sensors contribute to the differential regulation of activation and deactivation gating in these channels. A previously proposed model for S4 conformational changes during voltage-dependent activation can account for the differential regulation of gating seen in eag and HERG.     

Differences between ion binding to eag and HERG voltage sensors contribute to differential regulation of activation and deactivation gating

HERG (KCNH2) and ether-à-go-go (eag) (KCNH1) are members of the same subfamily of voltage-gated K+ channels. In eag, voltage-dependent activation is significantly slowed by extracellular divalent cations. To exert this effect, ions bind to a site located between transmembrane segments S2 and S3 in the voltage sensor domain where they interact with acidic residues that are conserved only among members of the eag subfamily. In HERG channels, extracellular divalent ions significantly accelerate deactivation. To investigate the ionbinding site in HERG, acidic residues in S2 and S3 were neutralized singly or in pairs to alanine, and the functional effects of extracellular Mg(2+) were characterized in Xenopus oocytes. To modulate deactivation kinetics in HERG, divalent cations interact with eag subfamily-specific acidic residues (D460 and D509) and also with an acidic residue in S2 (D456) that is widely conserved in the voltage-gated channel superfamily. In contrast, the analogous widely-conserved residue does not contribute to the ion-binding site that modulates activation kinetics in eag. We propose that structural differences between the ion-binding sites in the eag and HERG voltage sensors contribute to the differential regulation of activation and deactivation gating in these channels. A previously proposed model for S4 conformational changes during voltagedependent activation can account for the differential regulation of gating seen in eag and HERG.     

Molecular determinants of the hanatoxin binding in voltage-gated K+-channel drk1

The carboxyl terminus of S3 segment (S3(C)) in voltage-gated potassium channels was proposed to bear the binding site for gating modifier toxins like Hanatoxin and a helical secondary structural arrangement was suggested. Due to the lack of complete structure in high resolution for such a channel molecule, no further direct experimental data to elucidate the mechanism for their binding conformations could thus far be derived. In order to examine the putative three-dimensional structure of S3(C) and to illustrate the residues required for Hanatoxin binding, molecular simulation and docking were performed, based on the solution structure of Hanatoxin and the structural information from lysine-scanning results for S3(C) fragment. From our results, it is indicated that both hydrophobic and electrostatic interactions are utilized to stabilize the toxin binding. Detailed docking residues and appropriate orientation for binding regarding hydrophobic/-philic environments are also described. Compared with the functional data proposed by previous studies, the helical structural arrangement for the C-terminus of S3 segment in voltage-gated potassium channels can therefore be further emphasized.     

Changes in local S4 environment provide a voltage-sensing mechanism for mammalian hyperpolarization-activated HCN channels

The positively charged S4 transmembrane segment of voltage-gated channels is thought to function as the voltage sensor by moving charge through the membrane electric field in response to depolarization. Here we studied S4 movements in the mammalian HCN pacemaker channels. Unlike most voltage-gated channel family members that are activated by depolarization, HCN channels are activated by hyperpolarization. We determined the reactivity of the charged sulfhydryl-modifying reagent, MTSET, with substituted cysteine (Cys) residues along the HCN1 S4 segment. Using an HCN1 channel engineered to be MTS resistant except for the chosen S4 Cys substitution, we determined the reactivity of 12 S4 residues to external or internal MTSET application in either the closed or open state of the channel. Cys substitutions in the NH2-terminal half of S4 only reacted with external MTSET; the rates of reactivity were rapid, regardless of whether the channel was open or closed. In contrast, Cys substitutions in the COOH-terminal half of S4 selectively reacted with internal MTSET when the channel was open. In the open state, the boundary between externally and internally accessible residues was remarkably narrow (approximately 3 residues). This suggests that S4 lies in a water-filled gating canal with a very narrow barrier between the external and internal solutions, similar to depolarization-gated channels. However, the pattern of reactivity is incompatible with either classical gating models, which postulate a large translational or rotational movement of S4 within a gating canal, or with a recent model in which S4 forms a peripheral voltage-sensing paddle (with S3b) that moves within the lipid bilayer (the KvAP model). Rather, we suggest that voltage sensing is due to a rearrangement in transmembrane segments surrounding S4, leading to a collapse of an internal gating canal upon channel closure that alters the shape of the membrane field around a relatively static S4 segment.     

Substitution of a hydrophobic residue alters the conformational stability of Shaker K+ channels during gating and assembly.

A leucine residue at position 370 (L370) in 29-4 Shaker K+ channels resides within two overlapping sequence motifs conserved among most voltage-gated channels: the S4 segment and a leucine heptad repeat. Here we investigate the effects observed upon substitution of L370 with many other uncharged amino acid residues. We find that smaller or more hydrophilic residues produce greater alterations in both activation and inactivation gating than does substitution with other large hydrophobic residues. In addition, subunits containing less conservative substitutions at position 370 are restricted in their assembly with wild-type subunits and are unlikely to form homomultimeric channel complexes. Consistent with the idea that L370 influences the tertiary structure of these channels, the results indicate that L370 undergoes specific hydrophobic interactions during the conformational transitions of gating; similar interactions may take place during the folding, insertion, or assembly of Shaker K+ channel subunits.