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Ca²⁺ and cAMP-activated Cl^- conductances mediate Cl^- secretion in a mouse renal inner medullary collecting duct cell line

S. H. Boese, M. Glanville, O. Aziz, M. A. Gray and N. L. Simmons

1. The nature of Cl^- conductance(s) participating in transepithelial anion secretion by renal inner medullary collecting duct (IMCD, mIMCD-K2 cell line) was investigated.

2. Extracellular ATP (100 µM) stimulated a transient increase in both whole-cell Cl^- conductance and intracellular free Ca²⁺. In contrast, ionomycin (10-100 nM) caused a sustained increase in whole-cell Cl^- conductance. Pre-loading cells with the Ca²⁺ buffer BAPTA abolished the ATP-dependent responses and delayed the onset of the increase observed with ionomycin.

3. The Ca²⁺-activated whole-cell Cl^- current stimulated by ATP (peak) and ionomycin (maximal) displayed (i) a linear steady-state current-voltage relationship and (ii) time and voltage dependence with slow activation at +80 mV and slow inactivation at -80 mV. In BAPTA-loaded cells, ionomycin-elicited whole-cell currents exhibited pronounced outward rectification with time-dependent activation/inactivation.

4. Ca²⁺-activated and forskolin-activated Cl^- conductances co-exist since ATP activation of whole-cell current occurred during a maximal stimulation by forskolin in single cell recordings.

5. In IMCD epithelial layers, ATP and ionomycin stimulated an inward short circuit current (I_sc) dependent upon basal medium Na⁺ and Cl^-/HCO₃^- but independent of the presence of apical bathing medium Na⁺ and Cl^-/HCO₃^-. This was identical to forskolin stimulation and consistent with transepithelial anion secretion.

6. PCR amplification of reverse-transcribed mRNA using gene-specific primers demonstrated expression of both cystic fibrosis transmembrane conductance regulator (CFTR) mRNA and Ca²⁺-activated Cl^- channel (mCLCA1) mRNA in mIMCD-K2 cells.

7. Ca²⁺ and forskolin-activated Cl^- conductances participate in anion secretion by IMCD.

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