D. C. Devor

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 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.

Ion Channels and Transporters of Epithelia in Health and Disease

This book sheds new light on the physiology, molecular biology and pathophysiology of epithelial ion channels and transporters. It combines the basic cellular models and functions by means of a compelling clinical perspective, addressing aspects from the laboratory bench to the bedside. The individual chapters, written by leading scientists and clinicians, explore specific ion channels and transporters located in the epithelial tissues of the kidney, intestine, pancreas and respiratory tract, all of which play a crucial part in maintaining homeostasis. Further topics include the fundamentals of epithelial transport; mathematical modeling of ion transport; cell volume regulation; membrane protein folding and trafficking; transepithelial transport functions; and lastly, a discussion of transport proteins as potential pharmacological targets with a focus on the pharmacology of potassium channels

Characterization of a membrane potassium ion conductance in intestinal secretory cells using whole cell patch-clamp and calcium ion-sensitive dye techniques.

Publisher Summary This chapter describes a strategy for characterizing the changes in membrane K + conductance that result from exposure of isolated cells of the human secretory cell line T84 to a cholinergic agonist, and the role of intracellular Ca 2+ as a mediator of that process. These findings in isolated T84 cells are consistent with the proposed model for muscarinic agonist-induced Cl- secretion by an epithelium, in which an agonist-induced increase in basolateral membrane K + conductance hyperpolarizes the cells and causes secretion by increasing the driving force for C1- exit across the apical membrane. Care must be used in interpretation of results from dialyzed cells. This is illustrated in the studies by the difference seen between the duration of the carbachol-induced K + current oscillations and Ca 2+ oscillations. In every cell studied, the K + current returned to baseline in the continued presence of carbachol.