Actions of Ca2+ on an early stage in phototransduction revealed by the dynamic fall in Ca2+ concentration during the bright flash response

Abstract

To study the actions of Ca2+ on "early" stages of the transduction cascade, changes in cytoplasmic calcium concentration (Ca2+i) were opposed by manipulating Ca2+ fluxes across the rod outer segment membrane immediately following a bright flash. If the outer segment was exposed to 0 Ca2+/0 Na+ solution for a brief period immediately after the flash, then the period of response saturation was prolonged in comparison with that in Ringer solution. But if the exposure to 0 Ca2+/0Na+ solution instead came before or was delayed until 1 s after the flash then it had little effect. The degree of response prolongation increased with the duration of the exposure to 0 Ca2+/0 Na+ solution, revealing a time constant of 0.49 +/- 0.03 s. By the time the response begins to recover from saturation, Ca2+i seems likely to have fallen to a similar level in each case. Therefore the prolongation of the response when Ca2+i was prevented from changing immediately after the flash seems likely to reflect the abolition of actions of the usual dynamic fall in Ca2+i on an early stage in the transduction cascade at a site which is available for only a brief period after the flash. One possibility is that the observed time constant corresponds to the phosphorylation of photoisomerized rhodopsin.

Figure 1

Superimposed responses to bright flashes in Ringer solution and on exposure to 0 Ca²⁺/0 Na⁺ solution, (A) from 1 s before until 1 s after the flash, (B) from the time of the flash until 1 s after the flash, (C) from 1 s before the flash until the time of the flash, and (D) from 1 s after until 2 s after the flash. Each trace is the average of four responses; measurements in Ringer and 0 Ca²⁺/0 Na⁺ solution were bracketed symmetrically in time. Bright flash delivered 5,560 photons μm⁻².

Figure 2

Superimposed responses to bright flashes in Ringer solution and on exposure to 0 Ca²⁺/0 Na⁺ solution from 1 s before the flash until the times (Δt) after the flash indicated beside each trace, measured from the half-relaxation time of the junction current. Each trace is the average of four responses; measurements in Ringer solution and 0 Ca²⁺/0 Na⁺ solution were bracketed symmetrically in time. Bright flash delivered 5,350 photons μm⁻².

Figure 3

Collected data from 8 rods for the prolongation of the flash response by exposure to 0 Ca²⁺/0 Na⁺ solution as a function of the time spent in 0 Ca²⁺/0 Na⁺ solution after the flash, as in Fig. 2. Data represent the mean prolongation (mean ± SEM) of the time for 25% recovery of the original dark current plotted against the interval, Δt, between the flash and the half-relaxation time of the junction current on the return to Ringer solution. Values for Δt differed slightly between cells (total scatter ± 0.01 s), probably due to variation in the precise position of the interface between the two solution streams; mean values are plotted. Solid curve is a single exponential with time constant 0.49 ± 0.03 s offset from passing through the origin by 0.11 s, fitted using a weighted least squares algorithm. Bright flashes delivered 5,350 photons μm⁻².