Takushi Shimomura

Comparative Study of the Gating Motif and C-type Inactivation in Prokaryotic Voltage-gated Sodium Channels

Prokaryotic voltage-gated sodium channels (NaVs) 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 NaV, as well as of three new homologues cloned from Bacillus licheniformis (NaVBacL), Shewanella putrefaciens (NaVSheP), and Roseobacter denitrificans (NaVRosD). We found that, although activated by a lower membrane potential, NaVBacL inactivates as slowly as NaChBac. NaVSheP and NaVRosD 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 NaVs, but mutations in the regions changed the inactivation rates. Mutation of the region corresponding to the glycine hinge in NaVBacL (A214G), NaVSheP (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), NaVBacL (G224A), and NaVRosD (G217A) reduced the inactivation rate. These results imply that activation gating in prokaryotic NaVs does not require gating motifs and that the residues of helix S6 affect C-type inactivation rates in these channels.

Two Alternative Conformations of a Voltage-Gated Sodium Channel.

Activation and inactivation of voltage-gated sodium channels (Navs) are well studied, yet the molecular mechanisms governing channel gating in the membrane remain unknown. We present two conformations of a Nav from Caldalkalibacillus thermarum reconstituted into lipid bilayers in one crystal at 9Å resolution based on electron crystallography. Despite a voltage sensor arrangement identical with that in the activated form, we observed two distinct pore domain structures: a prominent form with a relatively open inner gate and a closed inner-gate conformation similar to the first prokaryotic Nav structure. Structural differences, together with mutational and electrophysiological analyses, indicated that widening of the inner gate was dependent on interactions among the S4-S5 linker, the N-terminal part of S5 and its adjoining part in S6, and on interhelical repulsion by a negatively charged C-terminal region subsequent to S6. Our findings suggest that these specific interactions result in two conformational structures.

Control of Spontaneous Ca2+ Transients Is Critical for Neuronal Maturation in the Developing Neocortex

Neural activity plays roles in the later stages of development of cortical excitatory neurons, including dendritic and axonal arborization, remodeling, and synaptogenesis. However, its role in earlier stages, such as migration and dendritogenesis, is less clear. Here we investigated roles of neural activity in the maturation of cortical neurons, using calcium imaging and expression of prokaryotic voltage-gated sodium channel, NaChBac. Calcium imaging experiments showed that postmigratory neurons in layer II/III exhibited more frequent spontaneous calcium transients than migrating neurons. To test whether such an increase of neural activity may promote neuronal maturation, we elevated the activity of migrating neurons by NaChBac expression. Elevation of neural activity impeded migration, and induced premature branching of the leading process before neurons arrived at layer II/III. Many NaChBac-expressing neurons in deep cortical layers were not attached to radial glial fibers, suggesting that these neurons had stopped migration. Morphological and immunohistochemical analyses suggested that branched leading processes of NaChBac-expressing neurons differentiated into dendrites. Our results suggest that developmental control of spontaneous calcium transients is critical for maturation of cortical excitatory neurons in vivo: keeping cellular excitability low is important for migration, and increasing spontaneous neural activity may stop migration and promote dendrite formation.

The C-terminal helical bundle of the tetrameric prokaryotic sodium channel accelerates the inactivation rate

Most tetrameric channels have cytosolic domains to regulate their functions, including channel inactivation. Here we show that the cytosolic C-terminal region of NavSulP, a prokaryotic voltage-gated sodium channel cloned from Sulfitobacter pontiacus, accelerates channel inactivation. The crystal structure of the C-terminal region of NavSulP grafted into the C-terminus of a NaK channel revealed that the NavSulP C-terminal region forms a four-helix bundle. Point mutations of the residues involved in the intersubunit interactions of the four-helix bundle destabilized the tetramer of the channel and reduced the inactivation rate. The four-helix bundle was directly connected to the inner helix of the pore domain, and a mutation increasing the rigidity of the inner helix also reduced the inactivation rate. These findings suggest that the NavSulP four-helix bundle has important roles not only in stabilizing the tetramer, but also in accelerating the inactivation rate, through promotion of the conformational change of the inner helix.

Arrangement and mobility of the voltage sensor domain in prokaryotic voltage-gated sodium channels

Prokaryotic voltage-gated sodium channels (NaVs) 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 (KVs), 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 NaVs remains limited. Here, we show that the domain arrangement in NaChBac, a firstly cloned prokaryotic NaV, is similar to that in KVs. 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 NaVs are similar to those in canonical KVs.

Evidence for lateral mobility of voltage sensors in prokaryotic voltage-gated sodium channels

Voltage-sensor domains (VSDs) in voltage-gated ion channels are thought to regulate the probability that a channel adopts an open conformation by moving vertically in the lipid bilayer. Here we characterized the movement of the VSDs of the prokaryotic voltage-gated sodium channel, NaChBac. Substitution of residue T110, which is located on the extracellular side of the fourth transmembrane helix of the VSD, by cysteine resulted in the formation of a disulfide bond between adjacent subunits in the channel. Our results suggest that T110 residues in VSDs of adjacent subunits can come into close proximity, implying that the VSDs can move laterally in the membrane and constitute a mechanism that regulates channel activity.

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

Voltage‐gated sodium channels are crucial for electro‐signalling in living systems. Analysis of the molecular mechanism requires both fine electrophysiological evaluation and high‐resolution channel structures. Here, we optimized a dual expression system of NavAb, which is a well‐established standard of prokaryotic voltage‐gated sodium channels, for E. coli and insect cells using a single plasmid vector to analyse high‐resolution protein structures and measure large ionic currents. Using this expression system, we evaluated the voltage dependence and determined the crystal structures of NavAb wild‐type and two mutants, E32Q and N49K, whose voltage dependence were positively shifted and essential interactions were lost in voltage sensor domain. The structural and functional comparison elucidated the molecular mechanisms of the voltage dependence of prokaryotic voltage‐gated sodium channels.

Molecular determinants of prokaryotic voltage‐gated sodium channels for recognition of local anesthetics

Local anesthetics (LAs) inhibit mammalian voltage‐gated Na+ channels (Navs) and are thus clinically important. LAs also inhibit prokaryotic Navs (BacNavs), which have a simpler structure than mammalian Navs. To elucidate the detailed mechanisms of LA inhibition to BacNavs, we used NavBh, a BacNav from Bacillus halodurans, to analyze the interactions of several LAs and quaternary ammoniums (QAs). Based on the chemical similarity of QA with the tertiary‐alkylamine (TAA) group of LAs, QAs were used to determine the residues required for the recognition of TAA by NavBh. We confirmed that two residues, Thr220 and Phe227, are important for LA binding; a methyl group of Thr220 is important for recognizing both QAs and LAs, whereas Phe227 is involved in holding blockers at the binding site. In addition, we found that NavBh holds blockers in a closed state, consistent with the large inner cavity observed in the crystal structures of BacNavs. These findings reveal the inhibition mechanism of LAs in NavBh, where the methyl group of Thr220 provides the main receptor site for the TAA group and the bulky phenyl group of Phe227 holds the blockers inside the large inner cavity. These two residues correspond to the two LA recognition residues in mammalian Navs, which suggests the relevance of the LA recognition between BacNavs and mammalian Navs.