Abstract
Purpose:
Sulfhydryl-binding and reducing agents affect the photoresponse of vertebrate rods in a manner that suggests action at multiple points in the phototransduction cascade. Thus, compounds containing thiol groups such as the amino acid, cysteine, could alter photoreceptor physiology by forming disulfide bonds. We probed for such effects on salamander photoreceptors using electrophysiological methods.
Methods:
Photoresponses were recorded from the inner segment of salamander rods and cones using the suction electrode method, while perfusing the outer segment (OS) with different cysteine analogs. D-cysteine, L-cysteine and D-cysteine with a methyl ester moiety were used to test for stereo-specificity and to ensure access to the interior of the OS, respectively. Dark-adapted and partially bleached cells were recorded to test for effects on visual pigment and apo-opsin. Patch clamp recordings of membranes excised from salamander rod OSs were carried out to clarify the effects on the cyclic nucleotide-gated (CNG) channel.
Results:
Both D- and L-cysteine increased circulating current and flash response size of dark-adapted rods, consistent with a higher fraction of CNG channels in the open state. Partially bleached cones exhibited a two-fold recovery of sensitivity, while recovery of sensitivity was less pronounced in partially bleached rods. The greater effect in cones suggested that cysteine might not easily enter the rod OS. Indeed, when D-cysteine methyl ester was given to partially bleached rods, there was a rapid increase in circulating current, followed by a slower rise in current and recovery of absolute sensitivity. The latter effect indicated relief of bleaching adaptation. Unexpectedly, flash response kinetics sped up for cells given the cysteine methyl ester. Patch clamp recording of rod OS membrane showed increased currents in the presence of D-cysteine, confirming a direct effect on the channels.
Conclusions:
These results indicate that cysteine can affect at least two components of the phototransduction cascade. First, cysteine opens more CNG channels, resulting in larger responses. Second, in partially bleached photoreceptors, cysteine appears to partially quench ongoing cascade activation, as shown by the recovery of sensitivity, possibly by suppressing the activity of opsin. The acceleration of response kinetics in partially bleached cells by cysteine methyl ester suggests that an additional cascade component may be affected.
Keywords: 648 photoreceptors •
715 signal transduction: pharmacology/physiology •
508 electrophysiology: non-clinical