Bradykinin-stimulated Cl- secretion in T84 cells. Role of Ca2+-activated hSK4-like K+ channels

Bradykinin (BK)-stimulated colonic Cl- secretion was studied in T84 colonic adenocarcinoma cells by measuring BK (50 nM)-evoked changes in cytosolic f ree [Ca2+] ([Ca2+](i)), membrane conductance and transepithelial ion transp ort. In T84 cells grown on impermeable supports, BK stimulated a transient increase in [Ca2+](i) as assessed by fura-2 ratio imaging. In cell-attached , patch-clamp recordings, BK transiently activated low-conductance K channe ls. These channels were activated/inactivated reversibly in inside-out patc hes by switching [Ca2+](i) in the bath between 30 nM and 100 nM. Excised ch annels recorded with 160 mM [K+] in bath and pipette exhibited an inwardly rectifying current/voltage-relation, conductances of 10 +/- 1 pS and 34 +/- 4 pS (n = 10) at positive and negative voltages, respectively, and a 15-fo ld lower permeability for Na+ than for K+. The mean open probability of the se channels did not depend on voltage but increased with increasing [Ca2+]( i) with an apparent concentration for a half-maximal response (EC50) Of 110 nM, resembling that of hSK4 K+ channels. Application of the reverse transc riptase-polymerase chain reaction technique showed hSK4 messenger ribonucle ic acid (mRNA) to be expressed in T84 cells. Macroscopic currents in T84 ce lls showed a similar dependence on [Ca2+](i). Whole cell conductance (in nS /10pF) increased from 0.5 +/- 0.1 (n = 6) at 10 nM [Ca2+](i) in the pipette solution to 1.5 +/- 0.2 (n = 7) at 100 nM, and to 2.0 +/- 0.5 (n = 7) at 1 mu M due to activation of a K+ conductance. In Ussing-chambered T84 monola yers grown on filters, BK did not evoke a short-circuit current (I-sc). Whe n, however, the monolayers were pre-stimulated by forskolin (1 mu M), BK fu rther enhanced Cl(-)secretion (Delta 1(sc) = 21 +/- 5 mu A/cm(2), n = 10) t ransiently and biphasically. In conclusion, BK enhances cy-die adenosine mo nophosphate-stimulated Cl- secretion in T84 cells, probably via basolateral , Ca2+-liganded activation of low-conductance hSK4-type K+ channels.