Identification of store-independent and store-operated Ca2+ conductances in Caenorhabditis elegans intestinal epithelial cells.
The nematode Caenorhabditis elegans offers significant experimental advantages for defining the genetic basis of diverse biological processes. Genetic and physiological analyses have demonstrated that inositol-1,4,5-trisphosphate (IP3)-dependent Ca2+ oscillations in intestinal epithelial cells play a central role in regulating the nematode defecation cycle, an ultradian rhythm with a periodicity of 45-50 s. Patch clamp studies combined with behavioral assays and forward and reverse genetic screening would provide a powerful approach for defining the molecular details of oscillatory Ca2+ signaling. However, electrophysiological characterization of the intestinal epithelium has not been possible because of its relative inaccessibility. We developed primary intestinal epithelial cell cultures that circumvent this problem. Intestinal cells express two highly Ca2+-selective, voltage-independent conductances. One conductance, IORCa, is constitutively active, exhibits strong outward rectification, is 60-70-fold more selective for Ca2+ than Na+, is inhibited by intracellular Mg2+ with a K1/2 of 692 microM, and is insensitive to Ca2+ store depletion. Inhibition of IORCa with high intracellular Mg2+ concentrations revealed the presence of a small amplitude conductance that was activated by passive depletion of intracellular Ca2+ stores. Active depletion of Ca2+ stores with IP3 or ionomycin increased the rate of current activation approximately 8- and approximately 22-fold compared with passive store depletion. The store-operated conductance, ISOC, exhibits strong inward rectification, and the channel is highly selective for Ca2+ over monovalent cations with a divalent cation selectivity sequence of Ca2+ > Ba2+ approximately Sr2+. Reversal potentials for ISOC could not be detected accurately between 0 and +80 mV, suggesting that PCa/PNa of the channel may exceed 1,000:1. Lanthanum, SKF 96365, and 2-APB inhibit both IORCa and ISOC reversibly. Our studies provide the first detailed electrophysiological characterization of voltage-independent Ca2+ conductances in C. elegans and form the foundation for ongoing genetic and molecular studies aimed at identifying the genes that encode the intestinal cell channels, for defining mechanisms of channel regulation and for defining their roles in oscillatory Ca2+ signaling.
Estevez
Ana Y
AY
Department of Anesthesiology, Vanderbilt University Medical Center, Nashville, TN 37232, USA.
Roberts
Randolph K
RK
Strange
Kevin
K
D000818:Animals; D017173:Caenorhabditis elegans; D002118:Calcium / Q000378:metabolism*; D015220:Calcium Channels / Q000378:metabolism*; D020013:Calcium Signaling / Q000502:physiology*; D002478:Cells, Cultured; D004553:Electric Conductivity; D004594:Electrophysiology; D004847:Epithelial Cells / Q000378:metabolism*; D015544:Inositol 1,4,5-Trisphosphate / Q000378:metabolism; D007413:Intestinal Mucosa / Q000378:metabolism; D007422:Intestines / Q000166:cytology; D007424:Intracellular Fluid / Q000378:metabolism*; D015640:Ion Channel Gating / Q000502:physiology; D007700:Kinetics; D008564:Membrane Potentials / Q000502:physiology; D018408:Patch-Clamp Techniques
D015220:Calcium Channels; D015544:Inositol 1,4,5-Trisphosphate; D002118:Calcium