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 (Palphabeta), and two gamma (Pgamma) subunits. In addition to catalytic sites, Palphabeta has two classes of noncatalytic cGMP binding sites with different affinities (Kd values <100 nM and >1 microM). Pgamma regulates Palphabeta as an inhibitor of cGMP hydrolysis and as a stimulator of cGMP binding to the high affinity noncatalytic sites. Pgamma release from Palphabeta by the GTP-bound alpha subunit of transducin (GTP.Talpha) interrupts these two functions. Here we describe a novel regulation of the Pgamma release by [cGMP] and its physiological implication. We isolated Pgamma mutants that exhibit abnormally one of these two functions, indicating the distinct domains in Pgamma are involved to express these functions. When [cGMP] was high ( approximately 5 microM), Pgamma responsible for the inhibition of cGMP hydrolysis was preferentially released, and cGMP hydrolysis activity of Palphabeta was increased about 10 times. When [cGMP] was low (less than approximately 0.5 microM), Pgamma responsible for the stimulation of cGMP binding to the high affinity sites was released. The Pgamma release resulted in the decrease of relative affinity of cGMP for the high affinity sites to at least (null)/1;10, followed by the rapid release of cGMP from one of the high affinity sites (apparent t1/2 = 3.8 s). cGMP ( approximately 5 microM) inhibited the extraction of Palphabeta from rod membranes by a Mg2+-free hypotonic buffer. The inhibition of Palphabeta extraction was not affected by Pgamma, suggesting that Palphabeta detects on the order of micromolar [cGMP] using low affinity noncatalytic sites on Palphabeta. Because [cGMP] is approximately 5 microM in darkness and lowered by photoexcitation and phosphodiesterase concentration is approximately 30 microM in rod photoreceptors, it is possible that cGMP phosphodiesterase functions to increase cytoplasamic [cGMP] after [cGMP] is reduced to the illuminated level.