Abstract
Thermal activation of the visual pigment constitutes a fundamental constraint on visual sensitivity. Its electrical correlate in the membrane current of dark-adapted rods are randomly occurring discrete "dark events" indistinguishable from responses to single photons. It has been proposed that thermal activation occurs in a small subpopulation of rhodopsin molecules where the Schiff base linking the chromophore to the protein part is unprotonated. On this hypothesis, rates of thermal activation should increase strongly with rising pH. The hypothesis has been tested by measuring the effect of pH changes on the frequency of discrete dark events in red rods of the common toad Bufo bufo. Dark noise was recorded from isolated rods using the suction pipette technique. Changes in cytoplasmic pH upon manipulations of extracellular pH were quantified by measuring, using fast single-cell microspectrophotometry, the pH-dependent metarhodopsin I-metarhodopsin II equilibrium and subsequent metarhodopsin III formation. These measurements show that, in the conditions of the electrophysiological experiments, changing perfusion pH from 6.5 to 9.3 resulted in a cytoplasmic pH shift from 7.6 to 8.5 that was readily sensed by the rhodopsin. This shift, which implies an 8-fold decrease in cytoplasmic [H(+)], did not increase the rate of dark events. The results contradict the hypothesis that thermal pigment activation depends on prior deprotonation of the Schiff base.