cAMP Increases Apical I_sK Channel Current and K⁺ Secretion in Vestibular Dark Cells

Abstract.

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 μA/cm² and from 4.3 ± 0.8 to 11.4 ± 2.2 pA, respectively. IBMX (1 mm) caused transient increases of I _sc from 415 ± 30 to 469 ± 38 μA/cm² and Ro-20-1724 (0.1 mm) from 565 ± 43 to 773 ± 58 μA/cm². 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.