Forty Years of Sodium Channels: Structure, Function, Pharmacology, and Epilepsy

Abstract

Voltage-gated sodium channels initiate action potentials in brain neurons. In the 1970s, much was known about the function of sodium channels from measurements of ionic currents using the voltage clamp method, but there was no information about the sodium channel molecules themselves. As a postdoctoral fellow and staff scientist at the National Institutes of Health, I developed neurotoxins as molecular probes of sodium channels in cultured neuroblastoma cells. During those years, Bruce Ransom and I crossed paths as members of the laboratories of Marshall Nirenberg and Philip Nelson and shared insights about sodium channels in neuroblastoma cells from my work and electrical excitability and synaptic transmission in cultured spinal cord neurons from Bruce's pioneering electrophysiological studies. When I established my laboratory at the University of Washington in 1977, my colleagues and I used those neurotoxins to identify the protein subunits of sodium channels, purify them, and reconstitute their ion conductance activity in pure form. Subsequent studies identified the molecular basis for the main functions of sodium channels-voltage-dependent activation, rapid and selective ion conductance, and fast inactivation. Bruce Ransom and I re-connected in the 1990s, as ski buddies at the Winter Conference on Brain Research and as faculty colleagues at the University of Washington when Bruce became our founding Chair of Neurology and provided visionary leadership of that department. In the past decade my work on sodium channels has evolved into structural biology. Molecular modeling and X-ray crystallographic studies have given new views of sodium channel function at atomic resolution. Sodium channels are also the molecular targets for genetic diseases, including Dravet Syndrome, an intractable pediatric epilepsy disorder with major co-morbidities of cognitive deficit, autistic-like behaviors, and premature death that is caused by loss-of-function mutations in the brain sodium channel Na_V1.1. Our work on a mouse genetic model of this disease has shown that its multi-faceted pathophysiology and co-morbidities derive from selective loss of electrical excitability and action potential firing in GABAergic inhibitory neurons, which disinhibits neural circuits throughout the brain and leads directly to the epilepsy, premature death and complex co-morbidities of this disease. It has been rewarding for me to use our developing knowledge of sodium channels to help understand the pathophysiology and to suggest potential therapeutic approaches for this devastating childhood disease.

Keywords: Epilepsy; Ion channel structure; Local anesthetics; Sodium channel.

Figure 1. Sodium channels as originally purified from mammalian brain

A. The α and β1 subunits of brain sodium channels analyzed by SDS-PAGE. B. Single channel currents from purified and reconstituted brain sodium channels. Sodium channels purified from rat brain were first reconstituted into phospholipid vesicles under conditions that yielded an average of one sodium channel molecule per vesicle. These vesicles fused with pre-formed planar phospholipid bilayers and sodium currents were recorded. Single channel currents with the voltage dependence and conductance of sodium channels in situ were recorded. C. A model of the purified rat brain sodium channel derived from biochemical experiments. This model depicts the state of the field in 1986 when it was drawn [32]. Since that time, cloning of sodium channel β subunits has revealed a family of four related genes, each of which encodes a single membrane-spanning protein with a large, glycosylated extracellular N-terminal domain composed primarily of an immunoglobulin-like fold and a small intracellular C-terminal domain [26, 27, 29].

Figure 2. Sodium channels in eukaryotes and prokaryotes

A. The α subunit of Na_V1.2 channels is illustrated as a transmembrane folding diagram in which cylinders represent transmembrane alpha helices and lines represent connecting amino acid sequences in proportion to their length. Structural components responsible for voltage sensing, pore formation, and fast inactivation are indicated. B. A low-resolution image of the sodium channel from electric eel electroplaque. C. A transmembrane folding diagram of the bacterial sodium channel NaChBac. Reprinted from [79].

Figure 3. A. Structure of the Na_VAb bacterial sodium channel at 2.7Å resolution

A. Top view. Colors represent temperature factors such that increased mobility in the crystals coded in warmer colors. Blue, pore-forming module; green to red, voltage-sensing module. B. Ion permeation pathway. The pore-forming modules of two Na_VAb subunits are shown surrounding the pore. S5, P helix, P2 helix, and S6 segments are labeled. Contour of the water-filled pore, gray. C. Three-dimensional structure of the voltage-sensing module of NavAb. Arginine gating charges, blue (R1–R4). Extracellular negative cluster (ENC), red; intracellular negative cluster (INC), red. Hydrophobic constriction site (HCS), green.

Figure 4. Drug receptor site in Na_VAb

A. Side view of Na_VAb with amino acid residues analogous to the Na_V1.2 drug receptor site colored. Pore portal denotes the fenestration leading to the membrane lipid phase in each of the four subunits of Na_VAb. B. Top view of Na_VAb colored as in A.