Deciphering voltage-gated Na(+) and Ca(2+) channels by studying prokaryotic ancestors

Abstract

Voltage-gated sodium channels (NaVs) and calcium channels (CaVs) are involved in electrical signaling, contraction, secretion, synaptic transmission, and other physiological processes activated in response to depolarization. Despite their physiological importance, the structures of these closely related proteins have remained elusive because of their size and complexity. Bacterial NaVs have structures analogous to a single domain of eukaryotic NaVs and CaVs and are their likely evolutionary ancestor. Here we review recent work that has led to new understanding of NaVs and CaVs through high-resolution structural studies of their prokaryotic ancestors. New insights into their voltage-dependent activation and inactivation, ion conductance, and ion selectivity provide realistic structural models for the function of these complex membrane proteins at the atomic level.

Keywords: Na(V)Ab; NaChBac; selectivity filter; slow inactivation; voltage sensor; voltage-gated calcium channel; voltage-gated sodium channel.

Figure 1. Na_V Channel Structure

(a) Two-dimensional schematic map of Na_V channel structure and function. 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. The roman numerals indicate the four homologous domains and the Arabic numerals are used to label the six transmembrane helices. The S4 helices are colored in red with “+” signs indicating gating charges. The S5-S5 helices are colored in green and the small white circles indicate key residues in the selectivity filter with “+” and “−“ signs indicating their charge states. The yellow circle with an “h” indicates inactivation gate. (b) Schematic map of the bacterial NaChBac channel, which contains the minimal functional elements of a single homologous domain in a mammalian Na_V channel.

Figure 2. Overall Structures of Prokaryotic Na_V Channels

(a) Structure of the Na_VAb bacterial Na_V channel determined at 2.7Å resolution and viewed from the extracellular side [9]. The four subunits of the homotetrameric channel are shown in different colors. The central pore is surrounded by four VSMs. (b) Side view of Na_VAb with the same coloring scheme as shown in (a). S5 and S6 helices of one subunit (slate) and the VSM of another subunit (cyan) are omitted for clarity. (c) Architecture of the Na_VAb pore with pore volume shown in grey and the high-strength-field (HSF) site residue Glu177 shown in sticks. P and P2 indicate the P and P2 helices. (d) Cross-section of the Na_VAb pore showing the closed activation gate. The selectivity filter is shown with electrostatic surface potential colored from −50 to 50 kT (red to blue). (e) Superposition of Na_VRh and Na_VAb with their PMs superimposed [9, 11]. The VSM of the two channels packs against the PM at different angles. (f) Structure of the Na_VMs pore module [13]. (g) Cross-section of the Na_VMs pore showing the open conformation of its activation gate [18]. The selectivity filter is shown with electrostatic surface potential colored from −50 to 50 kT (red to blue). (h) Structure of the Na_VAe1 pore with well-resolved C-terminal domain forming a four-helix bundle [15].

Figure 3. A Comparison of the Voltage-sensor Domains of Na_VAb and Na_VRh

The VSMs of Na_VAb (left, green) and Na_VRh (right, salmon) are shown in ribbon diagram [9, 11]. The sliding S4 helix is highlighted in yellow with the side chains of the four gating charge arginine residues (R1-R4) shown in sticks. Key residues forming the extracellular negative cluster (ENC), intracellular negative cluster (INC), and hydrophobic constriction site (HCS) are shown in sticks. The R4 residue of Na_VAb and Na_VRh interacts with the INC and ENC residues, respectively.

Figure 4. Structural Basis of Slow Inactivation

Specific structural features of Na_VAb-WT and Na_VRh, including a distorted selectivity filter and the breakdown of the four-fold symmetry of the pore, as expected for the slow inactivation state. Top panel: Close-up views of the extracellular entrance of Na_VAb-I217C (green, symmetric), Na_VAb-WT (grey, asymmetric), and Na_VRh (salmon, collapsed) with semi-transparent surface representation of the three channels [9-11]. The high-strength-field site glutamate residue in each structure along with its nearby serine residue is shown in sticks. Bottom panel: Close-up view of the intracellular closed activation gate of Na_VAb-I217C (symmetric, orange circle), Na_VAb-WT (asymmetric, orange oval), and Na_VRh (asymmetric, orange oval). .

Figure 5. Selectivity Filter of Na_VAb

(a) Close-up view of the extracellular entrance of Na_VAb-I217C with the high-strength-field site, Glu177, highlighted in yellow [9]. (b) Side view of the Na_VAb-I217C selectivity filter showing Glu177 (yellow) and the backbone carbonyls of Leu176 and Thr175, all of which are involved in selecting and permeating partially hydrated sodium ions. (c) Superposition of Na_VAb and a K⁺-channel (PDB code 1K4C) selectivity filter. The structural alignment, which is based on the common P-helices of the two channels, highlights the significant differences in the width of the two selectivity filters. (d) Different conformational states of the four high-strength-field site Glu177 residues captured in equilibrium molecular dynamics simulation of Na_VAb in a hydrated lipid bilayer with Na⁺ moving in and out of the pore [40]. The side chains of Glu177 point either out toward the mouth of the selectivity filter or into the lumen (0-4 indicate the number of Glu side chains dunked; Na⁺ ions not shown). (e) Axial distribution of Na⁺ atoms in the selectivity filter and central cavity of Na_VAb. Three distinguishable states are highlighted in which Na⁺ is directly bound to Glu177 (green), to both Glu177 and the backbone carbonyl of Leu176 (yellow), or to neither (brown). (d) and (e) are adapted from reference [40].

Figure 6. Ca²⁺ Selectivity and Permeation by Ca_VAb

(a) Side view of the superimposed selectivity filters of Ca_VAb and Na_VAb [9, 46]. The side chains of the three residues of Na_VAb mutated to generate Ca_VAb are colored in yellow in the Ca_VAb structure. The backbone structure of the selectivity filters of the two channels are nearly identical. (b) Side view of the Ca_VAb selectivity filter with three Ca²⁺ (green spheres) binding sites and their coordinating oxygen atoms. The distances between Ca²⁺ and the oxygen atoms indicated with dash lines range from 4.0 Å to 5.0 Å. (c) A proposed mechanism of Ca²⁺ permeation by Ca_VAb based on a combination of the “knock-out” and “stepwise permeation” models. The selectivity filter oscillates between two proposed ionic occupancy states where Ca²⁺ ions either bind to position 2, the high-affinity binding site, or position 1 and 3, which bind the ion at lower affinities.