Abstract
Prokaryotic voltage-gated sodium channels (Na(V)s) form homotetramers with each subunit contributing six transmembrane α-helices (S1-S6). Helices S5 and S6 form the ion-conducting pore, and helices S1-S4 function as the voltage sensor with helix S4 thought to be the essential element for voltage-dependent activation. Although the crystal structures have provided insight into voltage-gated K channels (K(V)s), revealing a characteristic domain arrangement in which the voltage sensor domain of one subunit is close to the pore domain of an adjacent subunit in the tetramer, the structural and functional information on Na(V)s remains limited. Here, we show that the domain arrangement in NaChBac, a firstly cloned prokaryotic Na(V), is similar to that in K(V)s. Cysteine substitutions of three residues in helix S4, Q107C, T110C, and R113C, effectively induced intersubunit disulfide bond formation with a cysteine introduced in helix S5, M164C, of the adjacent subunit. In addition, substituting two acidic residues with lysine, E43K and D60K, shifted the activation of the channel to more positive membrane potentials and consistently shifted the preferentially formed disulfide bond from T110C/M164C to Q107C/M164C. Because Gln-107 is located closer to the extracellular side of helix S4 than Thr-110, this finding suggests that the functional shift in the voltage dependence of activation is related to a restriction of the position of helix S4 in the lipid bilayer. The domain arrangement and vertical mobility of helix S4 in NaChBac indicate that the structure and the mechanism of voltage-dependent activation in prokaryotic Na(V)s are similar to those in canonical K(V)s.
Highlights
Prokaryotic NaVs are simpler than mammalian NaVs, comprising shorter polypeptides of ϳ300 amino acids that form homotetramers [3,4,5,6]
A previous study of Shaker KV showed that the cysteine substitutions of both Arg-362 in helix S4 and Ala-419 in helix S5 resulted in disulfide-bonded channel tetramers [12], indicating that residues Arg-362 and Ala-419 of the adjacent subunit are sufficiently close to each other to allow for the formation of a disulfide bond
The observation of disulfide-bonded tetramers for the T110C/M164C and R113C/M164C mutants indicates that residues Thr-110 and Arg-113 in helix S4 are close to residue Met-164 in helix S5 of the adjacent subunit (Fig. 2), suggesting that the putative domain arrangement shown in Fig. 1C is likely correct for prokaryotic NaVs
Summary
Using the same double cysteine mutagenesis approach previously used for the Shaker KV, we confirmed the proximity between helix S4 and helix S5 of an adjacent subunit in NaChBac. Helix S4 is thought to move vertically during voltage-dependent activation (18 –20). To examine the relationship between the mobility of helix S4 and voltage-dependent activation, we assessed the effect of mutations in helices S1 and S2, which shifted the activation of NaChBac to a more positive membrane potential. The additional mutations resulted in M164C forming disulfide bonds preferentially with residues in helix S4 closer to the extracellular surface. These results demonstrate that the vertical position of helix S4 depends on the charges surrounding the voltage sensor domain and that changes in the electrostatic environment shift the voltage dependence of activation of NaVs
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