The BK channel is a Ca²+- and voltage-gated potassium channel with many important physiological functions. To identify proteins important to its function in vivo, we screened for Caenorhabditis elegans mutants that suppressed a lethargic phenotype caused by expressing a gain-of-function (gf) isoform of the BK channel α-subunit SLO-1. BKIP-1 (for BK channel interacting protein), a small peptide with no significant homology to any previously characterized molecules, was thus identified. BKIP-1 and SLO-1 showed similar expression and subcellular localization patterns and appeared to interact physically through discrete domains. bkip-1 loss-of-function (lf) mutants phenocopied slo-1(lf) mutants in behavior and synaptic transmission and suppressed the lethargy, egg-laying defect, and deficient neurotransmitter release caused by SLO-1(gf). In heterologous expression systems, BKIP-1 decreased the activation rate and shifted the conductance-voltage relationship of SLO-1 in a Ca²+-dependent manner and increased SLO-1 surface expression. Thus, BKIP-1 is a novel auxiliary subunit critical to SLO-1 function in vivo.
C. elegans body-wall muscle cells are electrically coupled through gap junctions. Previous studies suggest that UNC-9 is an important, but not the only, innexin mediating the electrical coupling. Here we analyzed junctional current (Ij) for mutants of additional innexins to identify the remaining innexin(s) important to the coupling. The results suggest that a total of six innexins contribute to the coupling, including UNC-9, INX-1, INX-10, INX-11, INX-16, and INX-18. The Ij deficiency in each mutant was rescued completely by expressing the corresponding wild-type innexin specifically in muscle, suggesting that the innexins function cell-autonomously. Comparisons of Ij between various single, double, and triple mutants suggest that the six innexins probably form two distinct populations of gap junctions with one population consisting of UNC-9 and INX-18 and the other consisting of the remaining four innexins. Consistent with their roles in muscle electrical coupling, five of the six innexins showed punctate localization at muscle intercellular junctions when expressed as GFP- or epitope-tagged proteins, and muscle expression was detected for four of them when assessed by expressing GFP under the control of innexin promoters. The results may serve as a solid foundation for further explorations of structural and functional properties of gap junctions in C. elegans body-wall muscle.
Auxiliary subunits are often needed to tailor K+ channel functional properties and expression levels. Many auxiliary subunits have been identified for mammalian Slo1, a high-conductance K+ channel gated by voltage and Ca2+. Experiments with heterologous expression systems show that some of the identified Slo1 auxiliary subunits can also regulate other Slo K+ channels. However, it is unclear whether a single auxiliary subunit may regulate more than one Slo channel in native tissues. BKIP-1, an auxiliary subunit of C. elegans SLO-1, facilitates SLO-1 membrane trafficking and regulates SLO-1 function in neurons and muscle cells. Here we show that BKIP-1 also serves as an auxiliary subunit of C. elegans SLO-2, a high-conductance K+ channel gated by membrane voltage and cytosolic Cl- and Ca2+. Comparisons of whole-cell and single-channel SLO-2 currents in native neurons and muscle cells between worm strains with and without BKIP-1 suggest that BKIP-1 reduces chloride sensitivity, activation rate, and single-channel open probability of SLO-2. Bimolecular fluorescence complementation assays indicate that BKIP-1 interacts with SLO-2 carboxyl terminal. Thus, BKIP-1 may serve as an auxiliary subunit of SLO-2. BKIP-1 appears to be the first example that a single auxiliary subunit exerts opposite effects on evolutionarily related channels in the same cells.
The sinusoidal locomotion of Caenorhabditis elegans requires synchronous activities of neighboring body wall muscle cells. However, it is unknown whether the synchrony results from muscle electrical coupling or neural inputs. We analyzed the effects of mutating gap junction proteins and blocking neuromuscular transmission on the synchrony of action potentials (APs) and Ca2+ transients among neighboring body wall muscle cells. In wild-type worms, the percentage of synchronous APs between two neighboring cells varied depending on the anatomical relationship and junctional conductance (Gj) between them, and Ca2+ transients were synchronous among neighboring muscle cells. Compared with the wild type, knock-out of the gap junction gene unc-9 resulted in greatly reduced coupling coefficient and asynchronous APs and Ca2+ transients. Inhibition of unc-9 expression specifically in muscle by RNAi also reduced the synchrony of APs and Ca2+ transients, whereas expression of wild-type UNC-9 specifically in muscle rescued the synchrony defect. Loss of the stomatin-like protein UNC-1, which is a regulator of UNC-9-based gap junctions, similarly impaired muscle synchrony as unc-9 mutant did. The blockade of muscle ionotropic acetylcholine receptors by (+)-tubocurarine decreased the frequencies of APs and Ca2+ transients, whereas blockade of muscle GABAA receptors by gabazine had opposite effects. However, both APs and Ca2+ transients remained synchronous after the application of (+)-tubocurarine and/or gabazine. These observations suggest that gap junctions in C. elegans body wall muscle cells are responsible for synchronizing muscle APs and Ca2+ transients. The sinusoidal locomotion of Caenorhabditis elegans requires synchronous activities of neighboring body wall muscle cells. However, it is unknown whether the synchrony results from muscle electrical coupling or neural inputs. We analyzed the effects of mutating gap junction proteins and blocking neuromuscular transmission on the synchrony of action potentials (APs) and Ca2+ transients among neighboring body wall muscle cells. In wild-type worms, the percentage of synchronous APs between two neighboring cells varied depending on the anatomical relationship and junctional conductance (Gj) between them, and Ca2+ transients were synchronous among neighboring muscle cells. Compared with the wild type, knock-out of the gap junction gene unc-9 resulted in greatly reduced coupling coefficient and asynchronous APs and Ca2+ transients. Inhibition of unc-9 expression specifically in muscle by RNAi also reduced the synchrony of APs and Ca2+ transients, whereas expression of wild-type UNC-9 specifically in muscle rescued the synchrony defect. Loss of the stomatin-like protein UNC-1, which is a regulator of UNC-9-based gap junctions, similarly impaired muscle synchrony as unc-9 mutant did. The blockade of muscle ionotropic acetylcholine receptors by (+)-tubocurarine decreased the frequencies of APs and Ca2+ transients, whereas blockade of muscle GABAA receptors by gabazine had opposite effects. However, both APs and Ca2+ transients remained synchronous after the application of (+)-tubocurarine and/or gabazine. These observations suggest that gap junctions in C. elegans body wall muscle cells are responsible for synchronizing muscle APs and Ca2+ transients.
Chromium oxide, which has a lot of excellent properties, such as high hardness, low frictionfactor,so Chromium oxide films has good wear resistance and corrosion resistance. Chromium oxide filmswere deposited on silicon substrate by medium frequency unbalanced magnetron sputtering under differenttarget substrate spacing, sputtering power and bias voltage.The structure, composition, morphology and hardness of the chromium oxide films was studied by XRD,SEM, AFM, microhardness tester. Under optimized conditions, specifically, at target-substrate distance of100 mm and medium-frequency sputtering power of 11.2 kW, chromium oxide films with a single Cr2O3phase were acquired. The deposited coatings had a hardness of 20.5 GPa.
Invertebrate innexins and their mammalian homologues, the pannexins, are gap junction proteins. Although a large number of such proteins have been identified, few of the gap junctions that they form have been characterized to provide combined information of biophysical properties, coupling pattern, and molecular compositions. We adapted the dual whole cell voltage clamp technique to in situ analysis of electrical coupling in Caenorhabditis elegans body-wall muscle. We found that body-wall muscle cells were electrically coupled in a highly organized and specific pattern. The coupling was characterized by small (350 pS or less) junctional conductance (G(j)), which showed a bell-shaped relationship with junctional potential (V(j)) but was independent of membrane potential (V(m)). Injection of currents comparable to the junctional current (I(j)) into body-wall muscle cells caused significant depolarization, suggesting important functional relevance. The innexin UNC-9 appeared to be a key component of the gap junctions. Both Myc- and green fluorescent protein-tagged UNC-9 was localized to muscle intercellular junctions. G(j) was greatly inhibited in unc-9(fc16), a putative null mutant. Specific inhibition of UNC-9 function in muscle cells reduced locomotion velocity. Despite UNC-9 expression in both motor neurons and body-wall muscle cells, analyses of miniature and evoked postsynaptic currents in the unc-9 mutant showed normal neuromuscular transmission. These analyses provide a relatively detailed description of innexin-based gap junctions in a native tissue and suggest that innexin-based small conductance gap junctions can play an important role in processes such as locomotion.
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