Touran Zarrabi

A Molecular Switch between the Outer and the Inner Vestibules of the Voltage-gated Na+ Channel

Voltage-gated ion channels are transmembrane proteins that undergo complex conformational changes during their gating transitions. Both functional and structural data from K+ channels suggest that extracellular and intracellular parts of the pore communicate with each other via a trajectory of interacting amino acids. No crystal structures are available for voltage-gated Na+ channels, but functional data suggest a similar intramolecular communication involving the inner and outer vestibules. However, the mechanism of such communication is unknown. Here, we report that amino acid Ile-1575 in the middle of transmembrane segment 6 of domain IV (DIV-S6) in the adult rat skeletal muscle isoform of the voltage-gated sodium channel (rNaV1.4) may act as molecular switch allowing for interaction between outer and inner vestibules. Cysteine scanning mutagenesis of the internal part of DIV-S6 revealed that only mutations at site 1575 rescued the channel from a unique kinetic state (“ultra-slow inactivation,” IUS) produced by the mutation K1237E in the selectivity filter. A similar effect was seen with I1575A. Previously, we reported that conformational changes of both the internal and the external vestibule are involved in the generation of IUS. The fact that mutations at site 1575 modulate IUS produced by K1237E strongly suggests an interaction between these sites. Our data confirm a previously published molecular model in which Ile-1575 of DIV-S6 is in close proximity to Lys-1237 of the selectivity filter. Furthermore, these functional data define the position of the selectivity filter relative to the adjacent DIV-S6 segment within the ionic permeation pathway.

The Outer Vestibule of the Na+ Channel–Toxin Receptor and Modulator of Permeation as Well as Gating

The outer vestibule of voltage-gated Na+ channels is formed by extracellular loops connecting the S5 and S6 segments of all four domains (“P-loops”), which fold back into the membrane. Classically, this structure has been implicated in the control of ion permeation and in toxin blockage. However, conformational changes of the outer vestibule may also result in alterations in gating, as suggested by several P-loop mutations that gave rise to gating changes. Moreover, partial pore block by mutated toxins may reverse gating changes induced by mutations. Therefore, toxins that bind to the outer vestibule can be used to modulate channel gating.

Exploring the Structure of the Voltage-Gated Na+ Channel by an Engineered Drug Access Pathway to the Receptor Site for Local Anesthetics

Background: Currently the structure of eukaryotic voltage-gated Na+ channels (VGSCs) is predicted from available prokaryotic VGSC crystals. Results: In a mammalian VGSC, the predicted position of a multifunctional tryptophan in the external vestibule enabled design of a second conduction pathway. Conclusion: External parts of pro- and eukaryotic VGSCs are structurally similar. Significance: Engineered external drug access pathways may allow development of novel VGSC modulators. Despite the availability of several crystal structures of bacterial voltage-gated Na+ channels, the structure of eukaryotic Na+ channels is still undefined. We used predictions from available homology models and crystal structures to modulate an external access pathway for the membrane-impermeant local anesthetic derivative QX-222 into the internal vestibule of the mammalian rNaV1.4 channel. Potassium channel-based homology models predict amino acid Ile-1575 in domain IV segment 6 to be in close proximity to Lys-1237 of the domain III pore-loop selectivity filter. The mutation K1237E has been shown previously to increase the diameter of the selectivity filter. We found that an access pathway for external QX-222 created by mutations of Ile-1575 was abolished by the additional mutation K1237E, supporting the notion of a close spatial relationship between sites 1237 and 1575. Crystal structures of bacterial voltage-gated Na+ channels predict that the side chain of rNaV1.4 Trp-1531 of the domain IV pore-loop projects into the space between domain IV segment 6 and domain III pore-loop and, therefore, should obstruct the putative external access pathway. Indeed, mutations W1531A and W1531G allowed for exceptionally rapid access of QX-222. In addition, W1531G created a second non-selective ion-conducting pore, bypassing the outer vestibule but probably merging into the internal vestibule, allowing for control by the activation gate. These data suggest a strong structural similarity between bacterial and eukaryotic voltage-gated Na+ channels.

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.

A molecular switch between the outer and the inner vestibules of the voltage-gated Na+ channel

Background Voltage-gated ion channels are transmembrane proteins that undergo complex conformational changes during their gating transitions. Both functional and structural data from K channels suggest that extracellular and intracellular parts of the pore communicate with each other via a trajectory of interacting amino acids. No crystal structures are available for voltage-gated Na channels but functional data suggest a similar intramolecular communication involving the inner and outer vestibules. However, the mechanism of such communication is unknown. Here, we report that amino acid I1575 in the middle of transmembrane segment 6 of domain IV (DIV-S6) in the rNaV1.4 channel may act as molecular switch allowing for interaction between outer and inner vestibule.

The permanently charged lidocaine analogue QX222 acts as a blocker from the intracellular side and as an inactivation modulator from the extracellular side in a mutant NaV1.4 channel

Background QX222 is a quaternary amine analogue of lidocaine, which, unlike lidocaine, is permanently charged. Lidocaine has its binding site in the internal vestibule of the voltage-gated sodium channel. Due to the hydrophobic nature of its uncharged form, lidocaine reaches the binding site by passing through the membrane, QX222 can reach this binding site only by a hydrophilic pathway, presumably through the channel protein. However, such a pathway has been reported only in the heart-type sodium channel (NaV1.5) and some mutants of other sodium channels. Notably, mutations at site 1575 in the skeletal muscle-type sodium channel (NaV1.4) open an access pathway from the external side. In this study we tested the properties of QX222 block on the mutant I1575E.

Double mutant gating perturbation analysis predicts a high conformational stability of the domain IV S6 segment of the voltage-gated Na+ channel

The S6 segment of domain IV (DIV-S6) of the voltage-gated Na channel is considered to be a key player in gating and local anesthetic drug block. Thus, some mutations in DIV-S6 substantially alter the channels inactivation properties. In order to get a comprehensive picture of the kinetic role of DIV-S6 in fast inactivation we performed a cysteine scanning analysis of sites 1575-1591 in the DIV-S6 of the rNav1.4 channel. In addition, we produced the same cysteine replacements in the background of the mutation K1237E. K1237 is located in the P-loop of domain III and mutations at this site have dramatic effects both on permeation and gating properties. Hence, K1237E most likely causes a complex conformational change of the channel. We sought to explore whether K1237E changes the pattern of gating perturbations by the serial cysteine replacements in DIV-S6. The constructs were expressed in Xenopus laevis oocytes and studied by means of two electrode voltage-clamp. The half-point of availability following a 50 ms conditioning prepulse (V05) was -44 ± 1 mV and -51 ± 1 mV in wild-type and K1237E, respectively (P < 0.001) . Most serial amino acid replacements in DIV-S6 produced shifts in V05, both in wild-type and in K1237E background, ranging from +17 ± 1 mV to -9 ± 2 mV. A plot of the shifts in V05 by single DIV-S6 mutants relative to wild-type versus the shifts in V05 by double mutants relative to K1237E showed a significant positive correlation (R= 0.72, P=0.002). This indicates that the general pattern of gating perturbations in DIV-S6 is not affected by K1237E, suggesting a high conformational stability of the DIV-S6 segment during the fast inactivated state. Support: FWF P21006-B11

From frog oocytes to mammalian cells: substantial differences in modulation of NaV1.4 channel slow kinetic behaviour by the β1 subunit

Background Voltage gated sodium channels consist of an α subunit and several modulating β subunits. Upon depolarization, the α subunit first opens and then enters into different types of inactivated states. When expressed in mammalian cells, the β1 subunit has been shown to modulate the kinetics of fast inactivation. Here, we tested whether a very stable inactivated state, which we refer to as ultra-slow inactivation (Ius), is subject to modulation by the β1 subunit of the sodium channel. Previously, we showed that NaV1.4 channels, containing the mutation K1237E in the selectivity filter, had enhanced entry into Ius when expressed in Xenopus oocytes. Coexpression of the β1 subunit in this system had no effect on Ius. However, the kinetic behaviour of NaV1.4 may vary between the Xenopus oocyte system and mammalian expression systems. As both systems are widely used in ion channel research, it appeared of interest to evaluate the kinetic effect of coexpression of β1 in a mammalian expression system. Therefore, we tested whether Ius could be reproduced in TSA201 mammalian cells and whether it is subject to modulation by the β1 subunit in this system.

Interaction between the selectivity filter and the fast inactivation machinery in the voltage-gated Na+ channel

In the voltage-gated Na+ channel the central pore is believed to be lined by the S6 segments of all four domains. Conformational changes of these S6 segments are thought to give rise to channel opening, closing and fast inactivation (FI). Whereas FI most likely occurs by an occlusion of the intracellular part of the pore, the selectivity filter of the channel is located in the extracellular vestibule. We sought to investigate possible interactions between the selectivity filter and the intracellular part of the domain IV S6 segment which is known be involved in FI. To this end, a critical residue within the selectivity filter of the rNaV1.4 channel, K1237 was replaced by the negatively charged glutamate (K1237E). This mutation was combined with serial cysteine replacements of amino acids in the S6 segment of domain IV. In K1237E the midpoint of FI (V05) was shifted to the hyperpolarized direction relative to wild-type (-60 ± 13 vs. -47 ± 11 mV, n = 6, p < 0.01). Mutations of 16 residues in the domain IV-S6 produced inconsistent changes of V05. However, when these mutations were combined with K1237E, V05 was shifted to more negative values in all but one double mutants (mean shift -13 mV), irrespective of the direction and the amount of shift produced by the single S6 mutation. Conclusion: The selectivity filter of the voltage-gated Na+ channel is coupled to the machinery of FI.