Direct effect of Ca 2+ –calmodulin on cGMP‐activated Ca 2+ ‐dependent Cl − channels in rat mesenteric artery myocytes

Recently a novel cGMP‐activated Ca 2+ ‐dependent Cl − channel has been described in rat mesenteric artery smooth muscle cells. In the present work we have investigated the actions of calmodulin (CaM) on single channel cGMP‐activated Ca 2+ ‐dependent Cl − current ( I Cl(cGMP,Ca) ) in inside‐out patches. When 1 μ m CaM was applied to the intracellular surface of inside‐out patches bathed with 10 μ m cGMP and 100 n m [Ca 2+ ] i there was approximately a 10‐fold increase in channel open probability ( NP o ). This effect of CaM was not observed with lower [Ca 2+ ] i and 100 n m [Ca 2+ ] i with 1 μ m CaM did not activate Cl − channels in the absence of cGMP. The unitary conductance, reversal potential and mean open time of the single‐channel currents were similar in the absence or presence of CaM. With 10 μ m cGMP and 100 n m [Ca 2+ ] i the relationship between NP o and CaM concentration was well fitted by the Hill equation yielding an equilibrium constant for CaM of about 1.9 n m and a Hill coefficient of 1.7. With 1 μ m CaM (+10 μ m cGMP) the relationship between [Ca 2+ ] i and NP o was also fitted by the Hill equation which yielded an apparent equilibrium constant of 74 n m [Ca 2+ ] i and a Hill coefficient of 4.8. When [Ca 2+ ] i was increased from 300 n m to 1 μ m there was a decrease in NP o . The potentiating effect of CaM was markedly reduced by the selective CaM binding peptide Trp (5 n m ) but not by the Ca 2+ /CaM‐dependent protein kinase II (CaMKII) inhibitor autocamtide II related inhibitory peptide (AIP). It is concluded that CaM potentiates the activity of single channel I Cl(cGMP,Ca) by increasing the probability of channel opening via a CaMKII‐independent mechanism.