Proceedings of The Physiological Society
University of Manchester (2010) Proc Physiol Soc 19, C30
Effect of pH on the dominant time constant of salamander red-sensitive cone photoreceptors
J. Zang1, H. Matthews1
1. Physiology, Development & Neuroscience, University of Cambridge, Cambridge, United Kingdom.
Vertebrate phototransduction takes place via an enzymatic cascade in which photoisomerised photopigment acts via a G-protein to activate a phosphodiesterase which hydrolyses cGMP, closing cyclic nucleotide-gated channels in the outer segment membrane and causing the electrical response to light. The time constant dominating light response recovery is set by the slowest of the steps that quench the phototransduction cascade. In red-sensitive cones, the dominant time constant depends on [Ca2+]i (Matthews & Sampath, 2010), which declines during the light response and is known to modulate photopigment phosphorylation, and is accelerated by the removal of external Cl- ions (Matthews & Sampath, 2010), which interact with a binding site on the red cone photopigment (Kleinschmidt & Harosi, 1992), suggesting that pigment quenching dominates cone response recovery. To test this notion further, we investigated the effect on the dominant time constant of protons, which stabilise the active MII form of the photopigment (Parkes & Liebman, 1984). Red sensitive cones were isolated mechanically from the dark-adapted retina of aquatic Ambystoma tigrinum. The inner segment of an isolated cone was drawn into a suction pipette to record the photoresponse. The exposed outer segment was rapidly superfused for 6 or 9 s with a guanidinium-substituted 0Ca2+/0Na+ solution designed to minimize light-induced changes in [Ca2+]i (Matthews & Sampath, 2010), in which pH had been set to different values with an impermeant buffer. The dominant time constant was determined as the slope of the relationship between the time for 25% response recovery and the natural logarithm of the supersaturating flash intensity (Pepperberg et al. 1992). Acidification from the physiological value of pH 7.7 (49 cells) to pH 6.6 (9 cells) speeded the dominant time constant by a factor of 1.9 ± 0.3 (± SE), while alkalinisation slowed it by 1.5-fold (± 0.1) at pH 8.5 (12 cells) and 2.2-fold (± 0.2) at pH 9.0 (16 cells). The rate constants derived from these data could be fitted by a modified Henderson-Hasselbalch relationship with a pKa of 7.1 ± 0.1. Since [Ca2+]i was maintained at its dark value throughout the response, these changes represent actions of pH on response termination. As the photopigment is the only component of the cone transduction cascade exposed to external protons, which modify the proportion of rhodopsin in the active MII form which can be quenched (Mahalingam et al. 2008), these results strongly support the notion that photopigment quenching dominates red cone response recovery.
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