Structure and Function of the Voltage Sensor of Sodium Channels Probed by a β-Scorpion Toxin

Abstract

The voltage-gated ion channels and their structural relatives are a large superfamily of membrane proteins specialized for electrical signaling and ionic homeostasis (1). Voltage-gated sodium channels are responsible for the increase in sodium permeability that initiates action potentials in electrically excitable cells (2) and are the molecular target for several groups of neurotoxins, which bind to different receptor sites and alter voltage-dependent activation, conductance, and inactivation (3, 4). Sodium channels are composed of one pore-forming ␣ subunit of ϳ2000 amino acid residues associated with one or two smaller auxiliary subunits, ␤1-␤4 (5-7). The ␣ subunit consists of four homologous domains (I-IV), each containing six transmembrane segments (S1-S6), and a re-entrant pore loop (P) between S5 and S6 (5). The S4 transmembrane segments are positively charged and serve as voltage sensors to initiate channel activation (8 -14). However, the molecular mechanism of voltage sensing by sodium channels and the other members of the voltage-gated ion channel family is unknown.The initial "sliding helix" (9) or "helical screw" (15) models for voltage sensing proposed that the S4 segments, which have positively charged amino acids at intervals of three residues, transport gating charges outward to activate sodium channels in response to depolarization by moving along a spiral pathway through the protein structure. This movement would preserve interactions with surrounding hydrophilic and negatively charged amino acid residues during gating and thereby stabilize the gating charges in the intramembrane environment. Many structure-function studies have supported this general model (see "Discussion"). In contrast, x-ray crystallographic studies of a bacterial voltage-gated K ϩ channel in complex with detergent and a site-directed antibody yielded a structure in which the S3 and S4 segments lay along the position of the intracellular surface of the membrane, dissociated from the remainder of the protein (16 -18). These results led to the concept that the voltage sensors function as loosely linked "paddles," pivoting through the phospholipid surrounding the core of the ion channel as a semi-rigid body rather than moving gating charge outward through the protein structure. This paddle model makes strikingly different predictions for polypeptide toxins that modify gating by interaction with the voltage sensors. Whereas polypeptide toxins might be able to bind the extracellular end of the voltage sensors in the resting state in a sliding helix or helical screw gating model, the S4 segments would not be expected to be available for toxin binding in the resting state in the paddle model.Scorpion venoms contain two groups of polypeptides toxins that alter sodium channel gating. The ␣-scorpion toxins, as well as sea anemone toxins and some spider toxins, bind to neurotoxin receptor site 3 and slow or block inactivation (19 -22). Amino acid residues that contribute to neurotoxin receptor site

Solution Structure and Alanine Scan of a Spider Toxin That Affects the Activation of Mammalian Voltage-gated Sodium Channels

“…At least eight distinct toxin-binding sites have been characterized by radioligand binding studies on vertebrate and insect VGSCs (4,5). Among them, binding sites for polypeptide toxins from scorpions, sea anemones, spiders, and cone snails are distributed on the extracellular surface of the VGSC and have played a key role in the study of VGSC topology, function, and pharmacology (1,6,7). Scorpion ␣-toxins bind to receptor site 3, which involves extracellular loops of domains I and IV of the VGSC (8,9).…”

Mapping the Interaction Site for a β-Scorpion Toxin in the Pore Module of Domain III of Voltage-gated Na+ Channels

“…Mutagenesis-PCR-based site-directed mutagenesis was employed to generate single point mutations in WT rNa v 1.2 cDNA (9,11). Alanine-scanning mutagenesis was performed on the majority of the residues in IIISS2-S6 region.…”

“…cDNA Transfection-Detailed cDNA transfection procedures have been described elsewhere (9,11). cDNAs encoding WT or mutant rNa v 1.2a channels were co-transfected into tsA-201 cells with the marker protein pEBO-pCD8-leu2 using the calcium phosphate method.…”

“…Toxin derivatives can act as agonists or competitive antagonists of voltage-sensor trapping (10). Previous ligand binding (8,9) and electrophysiological studies (11) defined a molecular map of the receptor site for ␤-scorpion toxin IV of Centruroides suffusus suffusus (CssIV) 3 in the S1-S2 (IIS1-S2) and S3-S4 (IIS3-S4) extracellular loops of the voltage-sensing module in domain II. Structural studies of homotetrameric Na V and K V channels show that the S5-S6 loop in each subunit is in close proximity to the S1-S2 and S3-S4 loops of the adjacent subunit (3)(4)(5).…”

“…These effects work together to depolarize nerve and muscle fibers and cause conduction block. ␤-Scorpion toxins are thought to act via a voltage-sensor trapping mechanism in which the bound toxin binds and stabilizes the voltage sensor in domain II in its activated conformation (8,9). Toxin derivatives can act as agonists or competitive antagonists of voltage-sensor trapping (10).…”