Arrangement and Mobility of the Voltage Sensor Domain in Prokaryotic Voltage-gated Sodium Channels

Abstract

Prokaryotic voltage-gated sodium channels (Na V s) form homotetramers with each subunit contributing six transmembrane ␣-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.Voltage-gated ion channels play essential roles in electric signaling, muscle contraction, and other important physiologic processes (1). Mammalian voltage-gated sodium channels (Na V s) 2 are formed by a single, long polypeptide (ϳ2000 amino acids) that contains four homologous domains (2). Prokaryotic Na V s are simpler than mammalian Na V s, comprising shorter polypeptides of ϳ300 amino acids that form homotetramers (3-6). Each subunit, corresponding to one homologous domain in mammalian Na V s, contains six transmembrane ␣-helices (S1-S6). Helices S5 and S6 form the ionconducting pore in the center of the tetrameric channel, and helices S1-S4 form voltage sensors that surround the pore domain and detect the membrane potential. Helix S4 features a series of positively charged residues that are essential for voltage-dependent gating (7,8). It is thought that changes in the membrane potential cause some of these charges to move vertically in the lipid bilayer (9).NaChBac is a prokaryotic Na V cloned from Bacillus halodurans. Its function has been studied by expression in mammalian cells and confirmed to be a Na ϩ -selective channel (3), providing insight into gating charge movements related to voltage-dependent gating (10), and C-type inactivation (6, 11). Different prokaryotic Na V s differ in their voltage dependence and ion conduction kinetics (5, 6). The str...

Simplified Bacterial “Pore” Channel Provides Insight into the Assembly, Stability, and Structure of Sodium Channels

“…The sp-pore was tetrameric in the absence of the voltage sensor subdomain, which suggests that the association between monomers in the channel is largely mediated at the interfaces between the S5 and S6 transmembrane segments. Isolated voltage sensor domains do not assemble as tetramers (17, 18). It is therefore likely that the pore region (including its C terminus) is pivotal for forming the tetramers in the intact channels.…”

“…The pore subdomains form a compact central transmembrane pathway capable of accommodating ions in both selectivity and cavity regions; this is surrounded by the relatively loosely associated voltage sensor subdomains. Isolation and expression of the N-terminal four-transmembrane voltage sensor subdomain of the NaChBac channel (17) showed that the voltage sensor alone was capable of folding in the absence of the pore subdomain, and EPR measurements confirmed that it has a more tightly packed but similar overall fold to that of potassium voltage sensors (18). Here we designed a pore-only version of the Na v sp bacterial sodium channel and investigated whether it formed folded, stable tetramers and was capable of supporting Na + -specific ion channel permeability in the absence of the voltage sensor.…”

Optimized expression and purification of NavAb provide the structural insight into the voltage dependence

“…Maintaining the membrane potential at a more negative voltage than À180 mV for a long time, however, was very difficult. E43K and D60K of NaChBac, a BacNav homologue of Bacillus halodurans, are well known mutations that positively shift the voltage dependence of the activation [33]. In this protocol, run-down of the current of NavAb WT was not observed (Fig.…”

Structural physiology based on electron crystallography

“…High resolution images of radiation sensitive organic molecule, such as silver-7, 7 0 , 8,8 0 -tetracyanoquinondimethan complex, Ag-TCNQ, were easily and reproducibly observed by the MDS. 16 Unlike organic molecules, biological molecules are extremely radiation sensitive, and the resolution of their images is therefore strongly restricted by radiation damage.…”

“…6 Although no three-dimensional (3D-) structure has been determined yet for bacterial Na þ channels, mobility of voltage-sensor domains has been observed mainly by electrophysiology. 7,8 Simultaneously, we are studying water channels, which should be important for brain functions, because water content in brain is, remarkably, up to 85% and ion permeation has to be blocked to ensure the ion channel function, even when water molecules rapidly pass through the channels. A predominant water channel in brain is aquaporin-4 (AQP4).…”