Ian W. Glaaser

A Carboxyl-terminal Hydrophobic Interface Is Critical to Sodium Channel Function Relevance to Inherited Disorders

Perturbation of sodium channel inactivation, a finely tuned process that critically regulates the flow of sodium ions into excitable cells, is a common functional consequence of inherited mutations associated with epilepsy, skeletal muscle disease, autism, and cardiac arrhythmias. Understanding the structural basis of inactivation is key to understanding these disorders. Here we identify a novel role for a structural motif in the COOH terminus of the heart NaV1.5 sodium channel in determining channel inactivation. Structural modeling predicts an interhelical hydrophobic interface between paired EF hands in the proximal region of the NaV1.5 COOH terminus. The predicted interface is conserved among almost all EF hand-containing proteins and is the locus of a number of disease-associated mutations. Using the structural model as a guide, we provide biochemical and biophysical evidence that the structural integrity of this interface is necessary for proper Na+ channel inactivation gating. We thus demonstrate a novel role of the sodium channel COOH terminus structure in the control of channel inactivation and in pathologies caused by inherited mutations that disrupt it.

Interaction Between Fast and Ultra-Slow Inactivation in the Voltage-Gated Sodium Channel: Does the Inactivation Gate Stabilize the Channel Structure?

Recently, we reported that mutation A1529D in the domain (D) IV P-loop of the rat skeletal muscle Na+ channel μ1 (DIV-A1529D) enhanced entry to an inactivated state from which the channels recovered with an abnormally slow time constant on the order of ∼100 s. Transition to this “ultra-slow” inactivated state (USI) was substantially reduced by binding to the outer pore of a mutant μ-conotoxin GIIIA. This indicated that USI reflected a structural rearrangement of the outer channel vestibule and that binding to the pore of a peptide could stabilize the pore structure (Hilber, K., Sandtner, W., Kudlacek, O., Glaaser, I. W., Weisz, E., Kyle, J. W., French, R. J., Fozzard, H. A., Dudley, S. C., and Todt, H. (2001) J. Biol. Chem. 276, 27831–27839). Here, we tested the hypothesis that occlusion of the inner vestibule of the Na+ channel by the fast inactivation gate inhibits ultra-slow inactivation. Stabilization of the fast inactivated state (FI) by coexpression of the rat brain β1 subunit inXenopus oocytes significantly prolonged the time course of entry to the USI. A reduction in USI was also observed when the FI was stabilized in the absence of the β1 subunit, suggesting a causal relation between the occurrence of the FI and inhibition of USI. This finding was further confirmed in experiments where the FI was destabilized by introducing the mutations I1303Q/F1304Q/M1305Q. In DIV-A1529D + I1303Q/F1304Q/M1305Q channels, occurrence of USI was enhanced at strongly depolarized potentials and could not be prevented by coexpression of the β1 subunit. These results strongly suggest that FI inhibits USI in DIV-A1529D channels. Binding to the inner pore of the fast inactivation gate may stabilize the channel structure and thereby prevent USI. Some of the data have been published previously in abstract form (Hilber, K., Sandtner, W., Kudlacek, O., Singer, E., and Todt, H. (2002) Soc. Neurosci. Abstr. 27, program number 46.12).

Accessibility of mid-segment domain IV S6 residues of the voltage-gated Na+ channel to methanethiosulfonate reagents

The inner pore of the voltage‐gated Na+ channel is predicted by the structure of bacterial potassium channels to be lined with the four S6 α‐helical segments. Our previously published model of the closed pore based on the KcsA structure, and our new model of the open pore based on the MthK structure predict which residues in the mid‐portion of S6 face the pore. We produced cysteine mutants of the mid‐portion of domain IV‐S6 (Ile‐1575–Leu‐1591) in NaV1.4 and tested their accessibility to intracellularly and extracellularly placed positively charged methanethiosulfonate (MTS) reagents. We found that only two mutants, F1579C and V1583C, were accessible to both outside and inside 2‐(aminoethyl)‐methanethiosulfonate hydrobromide (MTSEA) Further study of those mutants showed that efficient closure of the fast inactivation gate prevented block by inside [2‐(trimethylammonium)ethyl]methanethiosulfonate bromide (MTSET) at slow stimulation rates. When fast inactivation was inhibited by exposure to anthropleurin B (ApB), increasing channel open time, both mutants were blocked by inside MTSET at a rate that depended on the amount of time the channel was open. Consistent with the fast inactivation gate limiting access to the pore, in the absence of ApB, inside MTSET produced block when the cells were stimulated at 5 or 20 Hz. We therefore suggest that the middle of IV‐S6 is an α‐helix, and we propose a model of the open channel, based on MthK, in which Phe‐1579 and Val‐1583 face the pore.

Involvement of local anesthetic binding sites on IVS6 of sodium channels in fast and slow inactivation

Local anesthetics (LAs) block Na(+) channels with a higher affinity for the fast or slow inactivated state of the channel. Their binding to the channel may stabilize fast inactivation or induce slow inactivation. We examined the role of the LA binding sites on domain IV, S6 (IVS6) of Na(+) channels in fast and slow inactivation by studying the gating properties of the mutants on IVS6 affecting LA binding. Mutation of the putative LA binding site, F1579C, inhibited fast and slow inactivation. Mutations of another putative LA binding site, Y1586C, and IVS6 residue involved in LA access and binding, I1575C, both enhanced fast and slow inactivation. None of the mutations affected channel activation. These results suggest that the LA binding site on IVS6 is involved in slow inactivation as well as fast inactivation, and these two gatings are coupled at the binding site.