A co-operative regulation of neuronal excitability by UNC-7 innexin and NCA/NALCN leak channel.

Gap junctions mediate the electrical coupling and intercellular communication between neighboring cells. Some gap junction proteins, namely connexins and pannexins in vertebrates, and innexins in invertebrates, may also function as hemichannels. A conserved NCA/Dmα1U/NALCN family cation leak channel regulates the excitability and activity of vertebrate and invertebrate neurons. In the present study, we describe a genetic and functional interaction between the innexin UNC-7 and the cation leak channel NCA in Caenorhabditis elegans neurons. While the loss of the neuronal NCA channel function leads to a reduced evoked postsynaptic current at neuromuscular junctions, a simultaneous loss of the UNC-7 function restores the evoked response. The expression of UNC-7 in neurons reverts the effect of the unc-7 mutation; moreover, the expression of UNC-7 mutant proteins that are predicted to be unable to form gap junctions also reverts this effect, suggesting that UNC-7 innexin regulates neuronal activity, in part, through gap junction-independent functions. We propose that, in addition to gap junction-mediated functions, UNC-7 innexin may also form hemichannels to regulate C. elegans' neuronal activity cooperatively with the NCA family leak channels.

Optogenetic analysis of synaptic function.

We introduce optogenetic investigation of neurotransmission (OptIoN) for time-resolved and quantitative assessment of synaptic function via behavioral and electrophysiological analyses. We photo-triggered release of acetylcholine or gamma-aminobutyric acid at Caenorhabditis elegans neuromuscular junctions using targeted expression of Chlamydomonas reinhardtii Channelrhodopsin-2. In intact Channelrhodopsin-2 transgenic worms, photostimulation instantly induced body elongation (for gamma-aminobutyric acid) or contraction (for acetylcholine), which we analyzed acutely, or during sustained activation with automated image analysis, to assess synaptic efficacy. In dissected worms, photostimulation evoked neurotransmitter-specific postsynaptic currents that could be triggered repeatedly and at various frequencies. Light-evoked behaviors and postsynaptic currents were significantly (P

A putative cation channel, NCA-1, and a novel protein, UNC-80, transmit neuronal activity in C. elegans.

Voltage-gated cation channels regulate neuronal excitability through selective ion flux. NALCN, a member of a protein family that is structurally related to the alpha1 subunits of voltage-gated sodium/calcium channels, was recently shown to regulate the resting membrane potentials by mediating sodium leak and the firing of mouse neurons. We identified a role for the Caenorhabditis elegans NALCN homologues NCA-1 and NCA-2 in the propagation of neuronal activity from cell bodies to synapses. Loss of NCA activities leads to reduced synaptic transmission at neuromuscular junctions and frequent halting in locomotion. In vivo calcium imaging experiments further indicate that while calcium influx in the cell bodies of egg-laying motorneurons is unaffected by altered NCA activity, synaptic calcium transients are significantly reduced in nca loss-of-function mutants and increased in nca gain-of-function mutants. NCA-1 localizes along axons and is enriched at nonsynaptic regions. Its localization and function depend on UNC-79, and UNC-80, a novel conserved protein that is also enriched at nonsynaptic regions. We propose that NCA-1 and UNC-80 regulate neuronal activity at least in part by transmitting depolarization signals to synapses in C. elegans neurons.

ITSN-1 controls vesicle recycling at the neuromuscular junction and functions in parallel with DAB-1.

Intersectins (Itsn) are conserved EH and SH3 domain containing adaptor proteins. In Drosophila melanogaster, ITSN is required to regulate synaptic morphology, to facilitate efficient synaptic vesicle recycling and for viability. Here, we report our genetic analysis of Caenorhabditis elegans intersectin. In contrast to Drosophila, C. elegans itsn-1 protein null mutants are viable and display grossly normal locomotion and development. However, motor neurons in these mutants show a dramatic increase in large irregular vesicles and accumulate membrane-associated vesicles at putative endocytic hotspots, approximately 300 nm from the presynaptic density. This defect occurs precisely where endogenous ITSN-1 protein localizes in wild-type animals and is associated with a significant reduction in synaptic vesicle number and reduced frequency of endogenous synaptic events at neuromuscular junctions (NMJs). ITSN-1 forms a stable complex with EHS-1 (Eps15) and is expressed at reduced levels in ehs-1 mutants. Thus, ITSN-1 together with EHS-1, coordinate vesicle recycling at C. elegans NMJs. We also found that both itsn-1 and ehs-1 mutants show poor viability and growth in a Disabled (dab-1) null mutant background. These results show for the first time that intersectin and Eps15 proteins function in the same genetic pathway, and appear to function synergistically with the clathrin-coat-associated sorting protein, Disabled, for viability.

A1152D mutation of the Na+ channel causes paramyotonia congenita and emphasizes the role of DIII/S4-S5 linker in fast inactivation.

Missense mutations in the human skeletal muscle Na+ channel alpha subunit (hSkM1) are responsible for a number of muscle excitability disorders. Among them, paramyotonia congenita (PC) is characterized by episodes of muscle stiffness induced by cold and aggravated by exercise. We have identified a new PC-associated mutation, which substitutes aspartic acid for a conserved alanine in the S4-S5 linker of domain III (A1152D). This residue is of particular interest since its homologue in the rat brain type II Na+ channel has been suggested as an essential receptor site for the fast inactivation particle. To identify the biophysical changes induced by the A1152D mutation, we stably expressed hSkM1 mutant or wild-type (WT) channels in HEK293 (human embryonic kidney) cells, and recorded whole-cell Na+ currents with the patch-clamp technique. Experiments were performed both at 21 and 11 degrees C to better understand the sensitivity to cold of paramyotonia. The A1152D mutation disrupted channel fast inactivation. In comparison to the WT, mutant channels inactivated with slower kinetics and displayed a 5 mV depolarizing shift in the voltage dependence of the steady-state. The other noticeable defect of A1152D mutant channels was an accelerated rate of deactivation from the inactivated state. Decreasing temperature by 10 degrees C amplified the differences in channel gating kinetics between mutant and WT, and unveiled differences in both the sustained current and channel deactivation from the open state. Overall, cold-exacerbated mutant defects may result in a sufficient excess of Na+ influx to produce repetitive firing and myotonia. In the light of previous reports, our data point to functional as well as phenotypic differences between mutations of conserved S4-S5 residues in domains II and III of the human skeletal muscle Na+ channel.

Functional characterization and cold sensitivity of T1313A, a new mutation of the skeletal muscle sodium channel causing paramyotonia congenita in humans.

Paramyotonia congenita (PC) is a dominantly inherited skeletal muscle disorder caused by missense mutations in the SCN4A gene encoding the pore-forming alpha subunit (hSkM1) of the skeletal muscle Na+ channel. Muscle stiffness is the predominant clinical symptom. It is usually induced by exposure to cold and is aggravated by exercise. The most prevalent PC mutations occur at T1313 on DIII-DIV linker, and at R1448 on DIV-S4 of the alpha subunit. Only one substitution has been described at T1313 (T1313M), whereas four distinct amino-acid substitutions were found at R1448 (R1448C/H/P/S). We report herein a novel mutation at position 1313 (T1313A) associated with a typical phenotype of PC. We stably expressed T1313A or wild-type (hSkM1) channels in HEK293 cells, and performed a detailed study on mutant channel gating defects using the whole-cell configuration of the patch-clamp technique. T1313A mutation impaired Na+ channel fast inactivation: it slowed and reduced the voltage sensitivity of the kinetics, accelerated the recovery, and decreased the voltage-dependence of the steady state. Slow inactivation was slightly enhanced by the T1313A mutation: the voltage dependence was shifted toward hyperpolarization and its steepness was reduced compared to wild-type. Deactivation from the open state assessed by the tail current decay was only slowed at positive potentials. This may be an indirect consequence of disrupted fast inactivation. Deactivation from the inactivation state was hastened. The T1313A mutation did not modify the temperature sensitivity of the Na+ channel per se. However, gating kinetics of the mutant channels were further slowed with cooling, and reached levels that may represent the threshold for myotonia. In conclusion, our results confirm the role of T1313 residue in Na+ channel fast inactivation, and unveil subtle changes in other gating processes that may influence the clinical phenotype.