Comparative study of the gating motif and C-type inactivation in prokaryotic voltage-gated sodium channels

Abstract

Prokaryotic voltage-gated sodium channels (Na(V)s) are homotetramers and are thought to inactivate through a single mechanism, named C-type inactivation. Here we report the voltage dependence and inactivation rate of the NaChBac channel from Bacillus halodurans, the first identified prokaryotic Na(V), as well as of three new homologues cloned from Bacillus licheniformis (Na(V)BacL), Shewanella putrefaciens (Na(V)SheP), and Roseobacter denitrificans (Na(V)RosD). We found that, although activated by a lower membrane potential, Na(V)BacL inactivates as slowly as NaChBac. Na(V)SheP and Na(V)RosD inactivate faster than NaChBac. Mutational analysis of helix S6 showed that residues corresponding to the "glycine hinge" and "PXP motif" in voltage-gated potassium channels are not obligatory for channel gating in these prokaryotic Na(V)s, but mutations in the regions changed the inactivation rates. Mutation of the region corresponding to the glycine hinge in Na(V)BacL (A214G), Na(V)SheP (A216G), and NaChBac (G219A) accelerated inactivation in these channels, whereas mutation of glycine to alanine in the lower part of helix S6 in NaChBac (G229A), Na(V)BacL (G224A), and Na(V)RosD (G217A) reduced the inactivation rate. These results imply that activation gating in prokaryotic Na(V)s does not require gating motifs and that the residues of helix S6 affect C-type inactivation rates in these channels.

FIGURE 1.

Phylogenetic tree of bacterial NaChBac homologues. A phylogenetic tree shows the bacterial species that express NaChBac homologues and their GenBank^TM accession numbers. The program ClustalW was used to calculate a multiple sequence alignment based on conserved sequences of the NaChBac homologues. The phylogenetic tree was generated using the program PROTDIST, part of the PHYLIP package (Phylogeny Inference Package; available on the World Wide Web). The branch lengths are proportional to the sequence divergence, with the scale bar corresponding to 0.1 substitution per amino acid site. Red underlined, homologues that were newly cloned and functionally characterized in this study; gray underlined, homologues that could not be cloned in this study; blue, homologues that were functionally characterized previously; gray, homologues that were previously cloned but did not show detectable channel activity.

FIGURE 2.

Primary structures and expression of NaChBac homologues. A, alignment of the deduced amino acid sequences of Na_VBacL, Na_VSheP, Na_VRosD, and NaChBac. The six putative transmembrane domains (S1–S6) are underlined in light gray, and the putative pore region is underlined in dark gray. Conserved arginines in helix S4 implicated in voltage sensing and residues constituting the putative sodium selectivity filter are shown as white letters in black boxes. The arrowheads indicate cleavage sites in Na_VSheP identified by MALDI mass spectrometry. B, Coomassie-stained SDS-PAGE of Na_VBacL, Na_VSheP, and Na_VRosD purified by Co²⁺ affinity chromatography. C, MALDI-TOF peptide mass spectrum of purified Na_VSheP. The x axis represents the mass-to-charge ratio (m/z), and the y axis represents relative abundance. The arrow indicates the full-length NaChBac homologues, and the arrowheads indicate fragments of Na_VSheP.

FIGURE 3.

Functional expression of Na_VBacL, Na_VSheP, and Na_VRosD in HEK 293 cells. A–C, representative traces of I_NaVBacL, I_NaVSheP, and I_NaVRosD resulting from the voltage protocol shown below (D–F). Averaged (n = 7; ±S.E.) peak current-voltage (I/V) relation of Na_VBacL, Na_VSheP, and Na_VRosD normalized by the peak current. V_Hold indicates the holding potential. The intersweep interval was 15 s.

FIGURE 4.

Voltage-dependent activation and inactivation of Na_VBacL, Na_VSheP, and Na_VRosD. A, I_NaVBacL deactivation tail currents. After prepulses of varying depolarization (from −120 to +30 mV, increments of +10 mV), tail currents were measured at −120 mV. B, I_NaVBacL steady-state inactivation currents. After a 2-s prepulse, the channels inactivated to a steady-state level and were reactivated by a second depolarizing pulse (−20 mV). C, I_NaVBacL-normalized activation curve (open circle, n = 9; ±S.E.) and steady-state inactivation curve (closed circle, n = 9; ±S.E.). D, I_NaVSheP deactivation tail currents. After prepulses of varying depolarization (from −120 to +30 mV, increments of +10 mV), tail currents were measured at −120 mV. E, I_NaVSheP steady-state inactivation currents. After a 500-ms prepulse, the channels inactivated to a steady-state level and were reactivated by a second depolarizing pulse (−20 mV). F, I_NaVSheP-normalized activation curve (open circle, n = 6; ±S.E.) and steady-state inactivation curve (closed circle, n = 6; ±S.E.). G, I_NaVRosD deactivation tail currents. After prepulses of varying depolarization (from −100 to +40 mV, increments of +10 mV), tail currents were measured at −120 mV. H, I_NaVRosD steady-state inactivation currents. After a 500-ms prepulse, the channels inactivated to a steady-state level and were reactivated by a second depolarizing pulse (+20 mV). I, I_NaVRosD-normalized activation curve (open circle, n = 6; ±S.E.) and steady-state inactivation curve (closed circle, n = 6; ±S.E.). The voltages with half-inactivation/activation and the slope factors are indicated as V_½ (mV) and κ (mV/e-fold), respectively in C, F, and I. The intersweep interval was 15 s in A, B, D, E, G, and H.

FIGURE 5.

Electrophysiological analysis of the effects of mutations in helix S6. A, alignment of helix S6 and inner helix sequences of Na_VBacL, Na_VSheP, Na_VRosD, Na_VSP, Na_VPZ, Na_VBP, NaChBac, KcsA (potassium channel from Streptomyces lividans), K_V1.2 (potassium channel from Rattus norvegicus), and the four subdomains (I–IV) of Na_V1.2 (voltage-gated sodium channel from rat brain). The arrowheads indicate the positions of G201 in Na_VBacL (blue), the glycine hinge (red), and the PXP motif (green); residues of these motifs are boxed in the corresponding colors. B and C, ribbon diagrams of KcsA (Protein Data Bank code 1r3j) (B) and K_V1.2 (Protein Data Bank code 2r9r) (C) with the residues and motifs described in A shown in the same colors. D, representative traces of currents produced by wild-type and mutant NaChBac channels activated by the following voltage protocol. After holding the potential at −120 mV, currents were measured at +10 mV for 500 ms (wild type (WT) and G219A/G229A), 100 ms (G219A), and 1 s (G229A). E, representative traces of currents produced by wild-type and mutant Na_VBacL channels activated by the following voltage protocol. After holding the potential at −160 mV, currents were measured at +10 mV for 500 ms (wild type, A214G, and A214G/G224A) and 1 s (G224A). F, representative traces of currents produced by wild-type and mutant Na_VSheP channels activated by the following voltage protocol. After holding the potential at −160 mV, currents were measured at −10 mV for 100 ms. G, representative traces of currents produced by wild-type and mutant Na_VRosD channels activated by the following voltage protocol. After holding the potential at −140 mV, currents were measured at +20 mV for 100 ms (wild type) and 300 ms (G217A and T207G/G217A).