Highly cooperative feedback control of retinal rod guanylate cyclase by calcium ions

Abstract

Visual excitation in retinal rod cells is mediated by a cascade that leads to the amplified hydrolysis of cyclic GMP (cGMP) and the consequent closure of cGMP-activated cation-specific channels in the plasma membrane^1–3. Recovery of the dark state requires the resynthesis of cGMP, which is catalysed by guanylate cyclase, an axoneme-associated enzyme^4–6. The lowering of the cytosolic calcium concentration (Ca_i) following illumination^7–10 is thought to be important in stimulating cyclase activity^11,12. This hypothesis is supported by the finding that the cGMP content of rod outer segments increases several-fold when Ca_i is lowered to less than 10 nM^13–16. It is evident that cGMP and Ca_i levels are reciprocally controlled by negative feedback^1,2. Guanylate cyclase from toad ROS is strongly stimulated when the calcium level is lowered from 10 μM to 10 nM, but only if they are excited by light¹⁷. We show here that the guanylate cyclase activity of unilluminated bovine rod outer segments increases markedly (5 to 20-fold) when the calcium level is lowered from 200 nM to 50 nM. This steep dependence of guanylate cyclase activity on the calcium level in the physiological range has a Hill coefficient of 3.9. Stimulation at low calcium levels is mediated by a protein that can be released from the outer segment membranes by washing with a low salt buffer. Calcium sensitivity is partially restored by adding the soluble extract back to the washed membranes. The highly cooperative activation of guanylate cyclase by the light-induced lowering of Ca_i is likely to be a key event in restoring the dark current after excitation.