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.

Exploring the structure of the voltage-gated Na+ channel by an engineered drug access pathway to the receptor site for local anesthetics.

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.

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 (rNa(V)1.4) 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," I(US)) 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 I(US). The fact that mutations at site 1575 modulate I(US) 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.

Speeding the recovery from ultraslow inactivation of voltage-gated Na+ channels by metal ion binding to the selectivity filter: a foot-on-the-door?

Slow inactivated states in voltage-gated ion channels can be modulated by binding molecules both to the outside and to the inside of the pore. Thus, external K(+) inhibits C-type inactivation in Shaker K(+) channels by a "foot-in-the-door" mechanism. Here, we explore the modulation of a very long-lived inactivated state, ultraslow inactivation (I(US)), by ligand binding to the outer vestibule in voltage-gated Na(+) channels. Blocking the outer vestibule by a mutant mu-conotoxin GIIIA substantially accelerated recovery from I(US). A similar effect was observed if Cd(2+) was bound to a cysteine engineered to the selectivity filter (K1237C). In K1237C channels, exposed to 30 microM Cd(2+), the time constant of recovery from I(US) was decreased from 145.0 +/- 10.2 s to 32.5 +/- 3.3 s (P < 0.001). Recovery from I(US) was only accelerated if Cd(2+) was added to the bath solution during recovery (V = -120 mV) from I(US), but not when the channels were selectively exposed to Cd(2+) during the development of I(US) (-20 mV). These data could be explained by a kinetic model in which Cd(2+) binds with high affinity to a slow inactivated state (I(S)), which is transiently occupied during recovery from I(US). A total of 50 microM Cd(2+) produced an approximately 8 mV hyperpolarizing shift of the steady-state inactivation curve of I(S), supporting this kinetic model. Binding of lidocaine to the internal vestibule significantly reduced the number of channels entering I(US), suggesting that I(US) is associated with a conformational change of the internal vestibule of the channel. We propose a molecular model in which slow inactivation (I(S)) occurs by a closure of the outer vestibule, whereas I(US) arises from a constriction of the internal vestibule produced by a widening of the selectivity filter region. Binding of Cd(2+) to C1237 promotes the closure of the selectivity filter region, thereby hastening recovery from I(US). Thus, Cd(2+) ions may act like a foot-on-the-door, kicking the I(S) gate to close.

C2C12 skeletal muscle cells adopt cardiac-like sodium current properties in a cardiac cell environment.

Intracardiac transplantation of undifferentiated skeletal muscle cells (myoblasts) has emerged as a promising therapy for myocardial infarct repair and is already undergoing clinical trials. The fact that cells originating from skeletal muscle have different electrophysiological properties than cardiomyocytes, however, may considerably limit the success of this therapy and, in addition, cause side effects. Indeed, a major problem observed after myoblast transplantation is the occurrence of ventricular arrhythmias. The most often transient nature of these arrhythmias may suggest that, once transplanted into cardiac tissue, skeletal muscle cells adopt more cardiac-like electrophysiological properties. To test whether a cardiac cell environment can indeed modify electrophysiological parameters of skeletal muscle cells, we treated mouse C(2)C(12) myocytes with medium preconditioned by primary cardiocytes and compared their functional sodium current properties with those of control cells. We found this treatment to significantly alter the activation and inactivation properties of sodium currents from "skeletal muscle" to more "cardiac"-like ones. Sodium currents of cardiac-conditioned cells showed a reduced sensitivity to block by tetrodotoxin. These findings and reverse transcription PCR experiments suggest that an upregulation of the expression of the cardiac sodium channel isoform Na(v)1.5 versus the skeletal muscle isoform Na(v)1.4 is responsible for the observed changes in sodium current function. We conclude that cardiomyocytes alter sodium channel isoform expression of skeletal muscle cells via a paracrine mechanism. Thereby, skeletal muscle cells with more cardiac-like sodium current properties are generated.

Selectivity filter residues contribute unequally to pore stabilization in voltage-gated sodium channels.

Mutations in the putative selectivity filter region of the voltage-gated Na+ channel, the so-called DEKA-motif, not only affect selectivity but also alter the channel's gating properties, suggesting functional coupling between permeation and gating. We have previously reported that charge-altering mutations at position 1237 in the P-loop of domain III (position K of the DEKA-motif in the adult rat skeletal muscle Na+ channel, rNa(v)1.4) dramatically enhanced entry to an inactivated state from which the channels recovered with a very slow time constant on the order of approximately 100 s (Todt, H., Dudley, S. C. J., Kyle, J. W., French, R. J., and Fozzard, H. A. (1999) Biophys. J. 76, 1335-1345). This state, termed "ultra-slow inactivation", may reflect a complex molecular rearrangement of the channel's pore region that involves both the extracellular and the cytoplasmic pore. Here, we tested whether charged DEKA-motif residues other than K1237 were also important determinants of a channel's gating properties. Therefore, we constructed the charge-neutralizing mutations D400A, E755A, and K1237A and studied the effects of these mutations on I(US). We found that, compared to wild-type rNa(v)1.4 channels, mutant D400A and K1237A but not E755A channels exhibited enhanced entry into ultra-slow inactivation. Selectivity for Na+ over K+, as judged from shifts in reversal potentials, was preserved in D400A, reduced in E755A, and completely lost in K1237A. These data suggest that an electrostatic interaction between the positively charged residue K1237 and the negatively charged residue D400 stabilizes the structure of the pore and thereby prevents I(US). Moreover, the interaction between K1237 and E755 appears to provide the basis for selective permeation of Na+ over K+.

Lidocaine: a foot in the door of the inner vestibule prevents ultra-slow inactivation of a voltage-gated sodium channel.

After opening, Na(+) channels may enter several kinetically distinct inactivated states. Whereas fast inactivation occurs by occlusion of the inner channel pore by the fast inactivation gate, the mechanistic basis of slower inactivated states is much less clear. We have recently suggested that the inner pore of the voltage-gated Na(+) channel may be involved in the process of ultra-slow inactivation (I(US)). The local anesthetic drug lidocaine is known to bind to the inner vestibule of the channel and to interact with slow inactivated states. We therefore sought to explore the effect of lidocaine binding on I(US). rNa(V) 1.4 channels carrying the mutation K1237E in the selectivity filter were driven into I(US) by long depolarizing pulses (-20 mV, 300 s). After repolarization to -120 mV, 53 +/- 5% of the channels recovered with a very slow time constant (tau(rec) = 171 +/- 19 s), typical for recovery from I(US). After exposure to 300 microM lidocaine, the fraction of channels recovering from I(US) was reduced to 13 +/- 4% (P < 0.01, n = 6). An additional mutation in the binding site of lidocaine (K1237E + F1579A) substantially reduced the effect of lidocaine on I(US), indicating that lidocaine has to bind to the inner vestibule of the channel to modulate I(US). We propose that I(US) involves a closure of the inner vestibule of the channel. Lidocaine may interfere with this pore motion by acting as a "foot in the door" in the inner vestibule.