A HIGHLY CALCIUM-SELECTIVE CATION CURRENT ACTIVATED BY INTRACELLULAR CALCIUM-RELEASE IN MDCK CELLS

1. The whole-cell patch clamp technique and fluorescence microscopy with the Ca2+ indicators fura-2 and fluo-3 were used to measure the whole-cell current and the free intracellular Ca2+ concentration ([Ca2+](i)) in Madin-Darby canine kidney (MDCK) cells. 2. In a Ca2+-free bath solution, thapsigargin (TG) caused a transient increase of [Ca2+](i). Subsequent addition of Ca2+ caused a long lasting elevation of [Ca2+](i). 3. In a Ca2+-free bath solution, extracellular application of TG, ATP or ionomycin, or intracellular application of inositol 1,4,5-trisphosphate (IP3), caused a small but significant inward current (I-in) and a transient outward Ca2+-dependent K+ current (I-K(Ca)), consistent with intracellular Ca2+ release. Subsequent addition of Ca2+ induced a prominent I-in with a current density of -4.2 +/- 0.7 pA pF(-1). This I-in was unaffected by inositol 1,3,4,5-tetrakisphosphate (IP3). 4. Na+ replacement by mannitol, N-methyl-D-glucamine(+) (NMG(+)), aminomethylidin-trimethanol(+) (Tris(+)) or choline(+) reduced I-in by 54, 65, 52 and 56%, respectively. This indicates an apparent Ca2+ selectivity over Na+ of 26:1. I-in was, however, unaffected by replacing Cl- with gluconate(-) or by the K+ channel blocker charybdotoxin (CTX). 5. I-in was completely blocked by La3+ (IC50 = 0.77 mu M). Consistently, La3+ completely reversed the TG-induced elevation of [Ca2+](i). SK&F 96365 (1-[3-(4-methoxyphenyl)-propoxyl]-1 -(4-methoxy-phenyl)-ethyl-1H-imidazole) HCl did not inhibit the TG-induced I-in. It did, however, exhibit a biphasic effect on [Ca2+](i), consisting of an initial Ca2+ decay and a subsequent Ca2+ elevation. La3+ completely reversed the SK&F 96365-induced elevation of [Ca2+](i). 6. In the absence of Na+, I-in was dependent on the bath Ca2+ concentration (EC(50) = 1.02 mM). Ca2+ replacement by Ba2+ or Mn2+ resulted in a reduction of I-in by 95 and 94%, respectively. From these experiments we conclude that Ca2+ release from intracellular Ca2+ stores, induced by different independent methods, stimulates La3+-inhibitable Ca2+ entry in MDCK cells. Ca2+ entry is at least, in part, mediated by a cation current, which is highly, but not exclusively, selective for Ca2+ over Na+ and insensitive to SK&F 96385.