Possible stimulation of retinal rod recovery to dark state by cGMP release from a cGMP phosphodiesterase noncatalytic site

Cyclic GMP phosphodiesterase, a key enzyme for phototransduction, contains alpha, beta (P alpha beta), and two gamma (P gamma) subunits, In addition to catalytic sites, P alpha beta has two classes of noncatalytic cGMP binding sites with different affinities (K-d values < 100 nM and >1 mu M). P gamma regulates P alpha beta as an inhibitor of cGMP hydrolysis and as a stimulator of cGMP binding to the high affinity noncatalytic sites. P gamma release from P alpha beta by the GTP-bound alpha subunit of transducin (GTP . T alpha) interrupts these two functions. Here we describe a novel regulation of the P gamma release by [cGMP] and its physiological implication, We isolated P gamma mutants that exhibit abnormally one of these two functions, indicating the distinct domains in P gamma are involved to express these functions, When [cGMP] was high (similar to 5 mu M), P gamma responsible for the inhibition of cGMP hydrolysis was preferentially released, and cGMP hydrolysis activity of P alpha beta was increased about 10 times, When [cGMP] was low (less than similar to 0.5 mu M), P gamma responsible for the stimulation of cGMP binding to the high affinity sites was released, The P gamma release resulted in the decrease of relative affinity of cGMP for the high affinity sites to at least 1/10, followed by the rapid release of cGMP from one of the high affinity sites (apparent t 1/2 = 3.8 s), cGMP (similar to 5 mu M) inhibited the extraction of P alpha beta from rod membranes by a Mg2+-free hypotonic buffer, The inhibition of P alpha beta extraction was not affected by P gamma, suggesting that P alpha beta detects on the order of micromolar [cGMP] using low affinity noncatalytic sites on P alpha beta. Because [cGMP] is similar to 5 mu M in darkness and lowered by photoexcitation and phosphodiesterase concentration is similar to 30 mu M in rod photoreceptors, it is possible that cGMP phosphodiesterase functions to increase cytoplasamic [cGMP] after [cGMP] is reduced to the illuminated level.