Two functionally distinct forms of guanosine cyclic 3',5'-phosphate stimulated cation channels in a bovine rod photoreceptor disk preparation

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Abstract
Cyclic nucleotide stimulated efflux of 22Na+ and 45Ca2+ from a purified bovine rod outer segment disk preparation was measured on the 25-100-ms time scale by a novel rapid superfusion method. Activation of cation efflux by 8-bromoguanosine cyclic 3'',5''-phosphate (8-Br-cGMP) was maximal within 25 ms. Over a wide range of concentrations of 8-Br-cGMP, the kinetics of termination of efflux precisely conformed to the sum of two exponential decay processes: a rapid phase (decay constant of 200 ms) and a slower phase (decay constant of 1.6 s). The kinetics of the biphasic decay of efflux cannot be explained by depletion of a pool of releaseable 22Na but appear to reflect an intrinsic process for inactivation of the channels. 8-Br-cGMP-stimulated release of actively accumulated 45Ca exhibited identical biphasic decay kinetics. The maximum rate of Ca release [5 nmol .cntdot.(mg of disk protein)-1 .cntdot. min-1] may be sufficient to produce a 1 .mu.M change in local cytoplasmic [Ca] within 20 ms. The Ca:Na selectivity ratio is .apprx.0.5:1 for both decay phases. 8-Br-cGMP demonstrated a lower potency (EC50 of 8.4 .mu.M vs 2.8 .mu.M) but a higher degree of cooperativity in its activation of the rapid vs the slower decay phase of 22Na efflux. The slower phase of decay was selectively inhibited by 25 .mu.M l-cis-diltiazem, a relatively weak inhibitor of the rapid decay phase. Sodium ion (5-10 mM) selectively inhibited the rapid decay phase of 8-Br-cGMP-stimulated 45Ca release. These two kinetically and pharmacologically distinct phases of decay are hypothesized to represent two functionally distinct forms of cGMP-stimulated cation channels.