Single cGMP-activated Ca2+-dependent Cl- channels in rat mesenteric artery smooth muscle cells

摘要

The present study describes the single channel properties of a novel cGMP-activated Ca2+-dependent Cl− channel in rat mesenteric artery smooth muscle cells. Single channel currents were recorded in cell-attached patches in the presence of 8Br cGMP in response to the addition of caffeine or noradrenaline and in both outside-out and inside-out patches when the internal patch surface was bathed in cGMP and Ca2+. The channels were permeable to Cl− ions with an anion permeability sequence of SCN− (1.7) > Cl− (1.0) > I− (0.6). Single channel mean open probability (NPo) was independent of voltage and the channels displayed three conductance levels of 15, 35 and 55 pS. cGMP was required for channel activation and the single channel NPo increased sharply with raised [Ca2+]i, maximal activation occurring at a [Ca2+]i of about 100 nm. The relationship between NPo and cGMP concentration was voltage independent and could be fitted by the Hill equation giving a Kd of about 3 μm and a Hill coefficient (nH) of 3. cGMP- and Ca2+-dependent channel currents were inhibited by 10 μm ZnCl2 but niflumic acid, an inhibitor of Ca2+-activated Cl− channels, had no effect. Inhibition of cGMP-dependent protein kinase activity by the cGMP-dependent protein kinase inhibitor KT5823 or replacement of ATP by AMP-PNP reduced NPo, while activation of cGMP-dependent protein kinase by guanosine 3′, 5′-cyclic monophosphate, β-phenyl-1, N2-etheno-8-bromo-sodium salt (8Br PET cGMP) produced a significant increase in single channel NPo. It is likely that these single channel currents underlie the noradrenaline-activated inward current important for vasomotion in these resistance arteries.