Incorporation of analogues of GTP and GDP into rod photoreceptors isolated from the tiger salamander

Abstract

1. Analogues of GTP and GDP were introduced into isolated rod photoreceptors using the whole-cell patch clamp technique, while simultaneously recording the photocurrent with a suction pipette. After several minutes of whole-cell recording the patch pipette was disengaged, thus trapping the analogue inside the cell. 2. During the introduction of the hydrolysis-resistant GTP analogues guanosine-5'-O-(3-thio-triphosphate) (GTP-gamma-S) and guanylyl-imidodiphosphate (GMP-PNP) the dark current progressively declined, and the duration of responses to flashes of light which had previously been just-saturating increased slightly. The form of the rising phases of the responses to dim or bright flashes was little affected. 3. Following the incorporation of these GTP analogues the response to an intense flash was prolonged by a factor of up to 300, and the circulating current remained suppressed for up to 1 h. Ultimately the circulating current recovered and the duration of the flash response returned to near its control value. 4. Superfusion of the outer segment with the phosphodiesterase inhibitor 3-isobutyl-1-methyl-xanthine (IBMX) during the extended period of saturation resulted in a rapid increase in the circulating current, suggesting that the analogues had their major effect on the duration of phosphodiesterase activation by light. 5. Introduction of the phosphorylation-resistant GDP analogue guanosine-5'-O-(2-thio-diphosphate) (GDP-beta-S) resulted in a decrease in light sensitivity and a reduction in the slope of the rising phase of the flash response. 6. The response to an intense flash was also prolonged in cells containing GDP-beta-S, recovery becoming progressively slower on successive presentations of the flash following the withdrawal of the patch pipette. This observation suggests that GDP-beta-S may be slowly converted within the cell to form a hydrolysis-resistant product. 7. These results indicate that the presence of a hydrolysis-resistant analogue of GTP within the cell causes light activation of the transduction mechanism for an extended period. Our interpretation of this finding is that hydrolysis of the bound guanosine nucleotide is necessary for the quenching of activated GTP-binding protein.