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

• Published in: The Journal of membrane biology Vol. 186; no. 3; pp. 145 - 157
• Main Authors: Izu, L T, McCulle, S L, Ferreri-Jacobia, M T, Devor, D C, Duffey, M E
• Format: Journal Article
• Language: English
• Published: United States Springer Nature B.V 01.04.2002
• Subjects: Caffeine - administration & dosage; Cell Line; Chlorides - metabolism; Cyclic AMP-Dependent Protein Kinases - administration & dosage; Electric Conductivity; Humans; Intestinal Mucosa - drug effects; Intestinal Mucosa - metabolism; Ion Transport; Membrane Potentials - drug effects; Patch-Clamp Techniques; Potassium Channels - metabolism; Sensitivity and Specificity; Temperature; Vasoactive Intestinal Peptide - administration & dosage
• ISSN: 0022-2631; 1432-1424
• DOI: 10.1007/s00232-001-0145-7

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 microA/cm2 to 75 +/- 11 microA/cm2 (n = 4), which was reduced to a near zero value by serosal applications of Ba2+ (5 mM). The chromanol, 293B (100 microM), 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.