Dual ion-channel regulation by cyclic GMP and cyclic GMP-dependent protein kinase.

Atrial natriuretic peptide, acting through its second messenger guanosine 3',5'-cyclic monophosphate (cGMP), suppresses Na+ absorption across the renal inner-medullary collecting duct and increases urinary Na+ excretion. Patch clamp studies show that cGMP reduces Na+ absorption by inhibiting an amiloride-sensitive cation channel in the apical membrane. We have now examined, using the patch clamp technique, the molecular mechanisms of cGMP inhibition. Cyclic GMP directly and specifically reduced the probability of a single channel being open (open probability, Po) by 39% (inhibition constant, Ki = 7.6 x 10(-7) M) by a phosphorylation-independent mechanism. Cyclic GMP also inhibited the channel by activating cGMP-dependent protein kinase (cGMP-kinase). Exogenous cGMP-kinase completely inhibited the channel by a phosphorylation-dependent mechanism. Activation of a pertussis toxin-sensitive G protein by GTP-gamma-S blocked cGMP-kinase inhibition of the channel. By contrast, cGMP-kinase inhibition of Po was completely reversed by GTP-gamma-S. Taken together with the results of a previous study showing that a G protein activates the cation channel, these data indicate that cGMP-kinase and a G protein sequentially regulate the cation channel. Our results show that atrial natriuretic peptide, acting through cGMP, inhibits Na+ absorption across the inner-medullary collecting duct by a dual mechanism, and that cGMP-kinase inhibits the channel by a pathway involving a G protein.     

Calcium-dependent regulation of cyclic GMP phosphodiesterase by a protein from frog retinal rods.

In vertebrate photoreceptors, light reduces cyclic GMP concentration and closes cGMP-activated channels to induce a hyperpolarizing response. As Ca2+ can permeate the channels and the Na(+)-Ca2+ exchanger continuously extrudes Ca2+, closure of the channel results in a reduction of the inter-rod Ca2+ concentration. This is believed to be one of the mechanisms of light-adaptation produced by activation of guanylate cyclase. Effects of Ca2+ on the cGMP phosphodiesterase (PDE) have been reported, but their physiological significance has remained unclear. We have perfused the inside-out preparation of a frog rod outer segment (I/O ROS, originally termed truncated ROS, and find that Ca2+ in a physiological range regulates the light-activation of PDE. Therefore, PDE regulation by Ca2+ must be involved in light-adaptation in rods. The effect is mediated by a newly found protein which binds to disk membranes at high Ca2+ concentrations and prolongs PDE activation.     

Induction by cyclic GMP of cationic conductance in plasma membrane of retinal rod outer segment

Vertebrate rod photoreceptors hyperpolarize when illuminated, due to the closing of cation-selective channels in the plasma membrane. The mechanism controlling the opening and closing of these channels is still unclear, however. Both 3',5'-cyclic GMP and Ca2+ ions have been proposed as intracellular messengers for coupling the light activation of the photopigment rhodopsin to channel activity and thus modulating light-sensitive conductance. We have now studied the effects of possible conductance modulators on excised 'inside-out' patches from the plasma membrane of the rod outer segment (ROS), and have found that cyclic GMP acting from the inner side of the membrane markedly increases the cationic conductance of such patches (EC50 30 microM cyclic GMP) in a reversible manner, while Ca2+ is ineffective. The cyclic GMP-induced conductance increase occurs in the absence of nucleoside triphosphates and, hence, is not mediated by protein phosphorylation, but seems rather to result from a direct action of cyclic GMP on the membrane. The effect of cyclic GMP is highly specific; cyclic AMP and 2',3'-cyclic GMP are completely ineffective when applied in millimolar concentrations. We were unable to recognize discrete current steps that might represent single-channel openings and closings modulated by cyclic GMP. Analysis of membrane current noise shows the elementary event to be 3 fA with 110 mM Na+ on both sides of the membrane at a membrane potential of -30 mV. If the initial event is assumed to be the closure of a single cyclic GMP-sensitive channel, this value corresponds to a single-channel conductance of 100 fS. It seems probable that the cyclic GMP-sensitive conductance is responsible for the generation of the rod photoresponse in vivo.     

Control of Ca2+ in rod outer segment disks by light and cyclic GMP

Photons absorbed in vertebrate rods and cones probably cause electrochemical changes at the photoreceptor plasma membrane by changing the cytoplasmic concentration of a diffusible transmitter substance, reducing the Na+ current flowing into the outer segment of the cell in the dark, to produce the observed membrane hyperpolarization that is the initial excitatory response. Cyclic GMP has been proposed as the transmitter because a light-activated cyclic GMP phosphodiesterase (PDE) has been found in rod disk membranes and because intracellularly injected cyclic GMP reduces rod membrane potentials. Free Ca2+ has also been proposed because increasing external [Ca2+] quickly and reversibly reduces the dark current and divalent cationophores increase the Ca2+ sensitivity. Ca2+ efflux from rod outer segments (ROS) of intact retinas occurs simultaneously with light responses. Vesicles prepared from ROS disk membranes become more permeable on illumination, releasing trapped ions or molecules, but intact outer segment disks have not previously been found to store sufficient Ca2+ in darkness and to release enough in light to meet the theoretical requirements for control of the dark current by varying cytoplasmic Ca2+ (refs 14-18). We now report experiments that show the required Ca2+ storage and release from rod disk membranes suspended in media containing high-energy phosphate esters and electrolytes approximating the cytoplasmic composition of live rod cells. Cyclic GMP stimulates Ca2+ uptake by ROS disks in such media.     

Opposite effects of cyclic GMP and cyclic AMP on Ca2+ current in single heart cells

The slow inward Ca2+ current, ICa, is fundamental in the initiation of cardiac contraction and neurohormonal regulation of cardiac function. It is increased by beta-adrenergic agonists, which stimulate synthesis of cyclic AMP (cAMP) and cAMP-dependent phosphorylation. The neurotransmitter acetylcholine reduces ICa by an unknown mechanism. There is strong evidence that acetylcholine reduces ICa by decreasing adenylate cyclase activity, but cGMP has also been implicated as ACh stimulates cGMP accumulation and activates cGMP-dependent protein kinase. Application of cGMP decreases contractile force, decreases Ca flux, shortens the duration of action potentials and inhibits Ca-dependent action potentials. Other studies, however, have concluded that cGMP levels do not correlate with contractile force and that cGMP has no effect on ICa. We have therefore examined the effects of intracellular perfusion of cGMP on ICa using isolated, voltage-clamped cells from frog ventricle. We find that cGMP has negligible effects on basal ICa, but greatly decreases the ICa that had been elevated by beta-adrenergic agonists or by intracellular perfusion with cAMP. The decrease of ICa is mediated by cAMP hydrolysis via a cGMP-stimulated cyclic nucleotide phosphodiesterase.