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Distinct calcium channel isoforms mediate parathyroid hormone and chlorothiazide-stimulated calcium entry in
1Department of Pharmacology and Toxicology, Dartmouth Medical School, Hanover, New Hampshire 03755-3835, USA.
Abstract:
Some cells express multiple calcium channel isoforms that are likely to have distinct functions. The present study used molecular cloning and antisense techniques to identify calcium channel isoforms mediating calcium entry in mouse distal convoluted tubule (DCT) cells. The DCT is the major site of hormone- and diuretic-regulated calcium transport in the kidney. Cellular calcium absorption involves entry through apical membrane calcium channels that are sensitive to dihydropyridine-type calcium channel antagonists. Partial cDNA clones corresponding to one isoform of the calcium channel alpha1 pore-forming subunit, alpha1C, and one isoform of the calcium channel beta accessory subunit, beta3, were isolated by RT-PCR. Full-length transcripts were detected by Northern blot analysis in immortalized DCT cells. Antisense oligonucleotides complementary to the alpha1C sequence inhibited the rise of intracellular calcium ([Ca2+]i) induced by the thiazide diuretic, chlorothiazide (CTZ), but not that induced by parathyroid hormone (PTH). However, antisense oligonucleotides complementary to the beta3 sequence inhibited both CTZ- and PTH-induced rises of [Ca2+]i. beta3 antisense oligonucleotides also inhibited the membrane hyperpolarization induced by CTZ but not that triggered by PTH. Thus, members of the voltage-gated calcium channel family are expressed in DCT cells, where they are responsible for hormone- and drug-induced calcium uptake. The results suggest that DCT cells contain multiple calcium channels with distinct roles in the regulation of cellular calcium.
Insights
Mouse kidney distal convoluted tubule cells utilize distinct calcium channels for hormone and diuretic regulation. This study identifies specific alpha1C and beta3 calcium channel subunits involved in cellular calcium uptake.
Area of Science:
- Nephrology
- Molecular Biology
- Cell Physiology
Background:
- Distal convoluted tubule (DCT) cells in the kidney regulate calcium transport.
- Hormones and diuretics influence DCT calcium absorption via apical membrane channels.
- Dihydropyridine-type calcium channel antagonists affect these channels.
Purpose of the Study:
- To identify calcium channel isoforms responsible for calcium entry in mouse DCT cells.
- To elucidate the distinct roles of identified calcium channel subunits in DCT calcium regulation.
Main Methods:
- Molecular cloning and RT-PCR to isolate partial cDNA clones for alpha1C and beta3 subunits.
- Northern blot analysis to detect full-length transcripts in immortalized DCT cells.
- Antisense oligonucleotide techniques to inhibit specific calcium channel subunit function.
Main Results:
- Alpha1C and beta3 calcium channel subunits were identified in mouse DCT cells.
- Antisense to alpha1C inhibited thiazide diuretic (chlorothiazide)-induced calcium rise, but not parathyroid hormone-induced rise.
- Antisense to beta3 inhibited both chlorothiazide- and parathyroid hormone-induced calcium rises and chlorothiazide-induced hyperpolarization.
Conclusions:
- Voltage-gated calcium channel family members are expressed in DCT cells.
- These channels mediate hormone- and drug-induced calcium uptake.
- Distinct calcium channels play specific roles in regulating cellular calcium in DCT cells.