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.Voltage-gated sodium channels (Na V s) 3 generate the rapid upstroke of action potentials in nerve cell axons (1). In mammalian Na V s, the channel is formed by the ␣-subunit, which comprises four repeats of six-transmembrane segments, with each repeat consisting of 300 -400 amino acids. The ␣-subunit carries several glycosylation sites and co-assembles with auxiliary subunits to form the native channel (2, 3). The only structural information on Na V s available to date is a density map of the Na V from the electric organ of the electric eel determined by cryoelectron microscopy (4). Due to its limited resolution of 19 Å, the density map did not provide insights into the gating or sodium selectivity.The first prokaryotic Na V , NaChBac, was cloned from Bacillus halodurans (5). Subsequently, three more prokaryotic sodium channels were cloned and characterized (6, 7). All studied prokaryotic Na V s form homotetramers with a structure thought to be similar to that of some potassium channels with known structures (8 -10). Furthermore, because the proteins could be expressed in large amounts in Escherichia coli and purified by metal chelate affinity chromatography (5, 7, 11), they are promising candidates for high resolution structure determination and structure-function analyses.The physiological role of prokaryotic Na V s may be related to pH homeostasis, motility, and chemotaxis (6, 12). Searching bacterial genomic data bases, we found 26 sequences of putative NaChBac homologues from bacteria living in various environments. We were able to clone the putative Na V genes from three of these bacteria, Bacillus licheniformis, Shewanella pu...

BackgroundPostsynaptic density (PSD)-95-like membrane-associated guanylate kinases (PSD-MAGUKs) are scaffold proteins in PSDs that cluster signaling molecules near NMDA receptors. PSD-MAGUKs share a common domain structure, including three PDZ (PDZ1/2/3) domains in their N-terminus. While multiple domains enable the PSD-MAGUKs to bind various ligands, the contribution of each PDZ domain to synaptic organization and function is not fully understood. Here, we focused on the PDZ1/2 domains of PSD-95 that bind NMDA-type receptors, and studied the specific roles of the ligand binding of these domains in the assembly of PSD proteins, synaptic properties of hippocampal neurons, and behavior, using ligand binding-deficient PSD-95 cDNA knockin (KI) mice.ResultsThe KI mice showed decreased accumulation of mutant PSD-95, PSD-93 and AMPA receptor subunits in the PSD fraction of the hippocampus. In the hippocampal CA1 region of young KI mice, basal synaptic efficacy was reduced and long-term potentiation (LTP) was enhanced with intact long-term depression. In adult KI mice, there was no significant change in the magnitude of LTP in CA1, but robustly enhanced LTP was induced at the medial perforant path-dentate gyrus synapses, suggesting that PSD-95 has an age- and subregion-dependent role. In a battery of behavioral tests, KI mice showed markedly abnormal anxiety-like behavior, impaired spatial reference and working memory, and impaired remote memory and pattern separation in fear conditioning test.ConclusionsThese findings reveal that PSD-95 including its ligand binding of the PDZ1/2 domains controls the synaptic clustering of PSD-MAGUKs and AMPA receptors, which may have an essential role in regulating hippocampal synaptic transmission, plasticity, and hippocampus-dependent behavior.

Voltage-dependent Ca2+ channels (Cavs) are indispensable for coupling action potentials with Ca2+ signaling in living organisms. The structure of Cavs is similar to that of voltage-dependent Na+ channels (Navs). It is known that prokaryotic Navs can obtain Ca2+ selectivity by negative charge mutations of the selectivity filter, but native prokaryotic Cavs had not yet been identified. We report the first identification of a native prokaryotic Cav, CavMr, whose selectivity filter contains a smaller number of negatively charged residues than that of artificial prokaryotic Cavs. A relative mutant whose selectivity filter was replaced with that of CavMr exhibits high Ca2+ selectivity. Mutational analyses revealed that the glycine residue of the CavMr selectivity filter is a determinant for Ca2+ selectivity. This glycine residue is well conserved among subdomains I and III of eukaryotic Cavs. These findings provide new insight into the Ca2+ selectivity mechanism that is conserved from prokaryotes to eukaryotes.

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...