cAMP increases apical I-sK channel current and K+ secretion in vestibular dark cells

Adenosine 3',5'-cyclic monophosphate (cAMP) is known to stimulate exogenous I-sK channel current in the Xenopus oocyte expression system. The present study was performed to determine whether elevation of cytosolic cAMP in a native mammalian epithelium known to secrete K+ through endogenously expressed I-sK channels would stimulate K+ secretion through these channels. The equivalent short circuit current (I-sc) across vestibular dark cell epithelium in gerbil was measured in a micro-Ussing chamber and the apical membrane current (I-IsK) and conductance (g(IsK)) of I-sK channels was recorded with both the on-cell macro-patch and nystatin-perforated whole-cell patch-clamp techniques. It has previously been shown that I-sc can be accounted for by transepithelial K+ secretion and that the apical I-sK channels constitute a significant pathway for K+ secretion. The identification of the voltage-dependent whole-cell currents in vestibular dark cells was strengthened by the finding that a potent blocker of I-sK channels, chromanol 293B, strongly reduced I-IsK from 646 +/- 200 to 154 +/- 22 pA (71%) and g(IsK) from 7.5 +/- 2.6 to 2.8 +/- 0.4 nS (53%). Cytoplasmic cAMP was elevated by applying dibutyryl cyclic AMP (dbcAMP), or the phosphodiesterase inhibitors 3-isobutyl-1-methylxanthine (IBMX) and Ro-20-1724. dbcAMP (1 mM) increased I-sc and I-IsK from 410 +/- 38 to 534 +/- 40 mu A/cm(2) and from 4.3 +/- 0.8 to 11.4 +/- 2.2 pA, respectively. IBMX (1 mM) caused transient increases of I,, from 415 +/- 30 to 469 +/- 38 mu A/cm(2) and Ro-20-1724 (0.1 mM) from 565 +/- 43 to 773 +/- 58 mu A/cm(2). IBMX increased I-IsK from 5.5 +/- 1.5 to 16.9 +/- 5.8 pA in on-cell experiments and from 191 +/- 31 to 426 +/- 53 pA in whole-cell experiments. The leak conductance due to all non-I-sK channel sources did not change during dbcAMP and IBMX while 293B in the presence of dbcAMP reduced I-Isk by 84% and g(IsK) by 62%, similar to unstimulated conditions. These results demonstrate that the cAMP pathway is constitutively active in vestibular dark cells and that the cAMP pathway stimulates transepithelial K+ secretion by increasing I-sK channel current rather than by altering another transport pathway.