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, I_ORCa, is constitutively active, exhibits strong outward rectification, is 60–70-fold more selective for Ca²⁺ than Na⁺, is inhibited by intracellular Mg²⁺ with a K_1/2 of 692 μM, and is insensitive to Ca²⁺ store depletion. Inhibition of I_ORCa 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 ∼8- and ∼22-fold compared with passive store depletion. The store-operated conductance, I_SOC, exhibits strong inward rectification, and the channel is highly selective for Ca²⁺ over monovalent cations with a divalent cation selectivity sequence of Ca²⁺ > Ba²⁺ ≈ Sr²⁺. Reversal potentials for I_SOC could not be detected accurately between 0 and +80 mV, suggesting that P_Ca/P_Na of the channel may exceed 1,000:1. Lanthanum, SKF 96365, and 2-APB inhibit both I_ORCa and I_SOC 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.