cAMP increases apical IsK channel current and K+ secretion in vestibular dark cells

Adenosine 3',5'-cyclic monophosphate (cAMP) is known to stimulate exogenous IsK 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 IsK channels would stimulate K+ secretion through these channels. The equivalent short circuit current (Isc) across vestibular dark cell epithelium in gerbil was measured in a micro-Ussing chamber and the apical membrane current (IsK) and conductance (gIsK) of IsK channels was recorded with both the on-cell macro-patch and nystatin-perforated whole-cell patch-clamp techniques. It has previously been shown that Isc can be accounted for by transepithelial K+ secretion and that the apical IsK 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 IsK channels, chromanol 293B, strongly reduced IIsK from 646 +/- 200 to 154 +/- 22 pA (71%) and gIsK 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 Isc and IIsK from 410 +/- 38 to 534 +/- 40 microA/cm2 and from 4.3 +/- 0.8 to 11.4 +/- 2.2 pA, respectively. IBMX (1 mM) caused transient increases of Isc from 415 +/- 30 to 469 +/- 38 microA/cm2 and Ro-20-1724 (0.1 mM) from 565 +/- 43 to 773 +/- 58 microA/cm2. IBMX increased IIsK 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-IsK channel sources did not change during dbcAMP and IBMX while 293B in the presence of dbcAMP reduced IIsK by 84% and gIsK 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 IsK channel current rather than by altering another transport pathway.