Vasoactive intestinal peptide-stimulated Cl^- secretion: Activation of cAMP-dependent K⁺ channels

Vasoactive intestinal peptide (VIP) stimulates active Cl^- secretion by the intestinal epithelium, a process that depends upon the maintenance of a favorable electrical driving force established by a basolateral membrane K⁺ conductance. To demonstrate the role of this K⁺ conductance, we measured short-circuit current (I_SC) across monolayers of the human colonic secretory cell line, T84. The serosal application of VIP (50 nM) increased I_SC from 3 ± 0.4 μA/cm² to 75 ± 11 μA/cm² (n = 4), which was reduced to a near zero value by serosal applications of Ba²⁺ (5 mM). The chromanol, 293B (100 μM), reduced I_SC by 74%, but charybdotoxin (CTX, 50 nM) had no effect. We used the whole-cell voltage-clamp technique to determine whether the K⁺ conductance is regulated by cAMP-dependent phosphorylation in isolated cells. VIP (300 nM) activated K⁺ current (131 ± 26 pA, n = 15) when membrane potential was held at the Cl^- equilibrium potential (E_Cl- = -2 mV), and activated inward current (179 ± 28 pA, n = 15) when membrane potential was held at the K⁺ equilibrium potential (E_K+ = -80 mV); however, when the cAMP-dependent kinase (PKA) inhibitor, PKI (100 nM), was added to patch pipettes, VIP failed to stimulate these currents. Barium (Ba²⁺, 5 mM), but not 293B, blocked this K⁺ conductance in single cells. We used the cell-attached membrane patch under conditions that favor K⁺ current flow to demonstrate the channels that underlie this K⁺ conductance. VIP activated inwardly rectifying channel currents in this configuration. Additionally, we used fura-2AM to show that VIP does not alter the intracellular Ca²⁺ concentration, [Ca²⁺]_i. Caffeine (5 mM), a phosphodiesterase inhibitor, also stimulated K⁺ current (185 ± 56 pA, n = 8) without altering [Ca²⁺]_i. These results demonstrate that VIP activates a basolateral membrane K⁺ conductance in T84 cells that is regulated by cAMP-dependent phosphorylation.