Incorporation of calcium buffers into salamander retinal rods: a rejection of the calcium hypothesis of phototransduction.

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Abstract
The suction-electrode technique was used to monitor the photocurrent of isolated retinal rods from the tiger salamander, by drawing in the light-sensitive outer segment, or sometimes the inner segment. Calcium buffers or other agents were then introduced into the rod cytoplasm by the whole-cell patch-clamp technique. A patch pipette was sealed against the region of the rod protruding from the suction pipette (usually the inner segment), and the membrane patch was ruptured to obtain a whole-cell recording. Several lines of evidence indicated that the pipette contents diffused into the outer segment, and showed that the cell could be adequately voltage clamped. With only trace quantities of chelator in the patch pipette (to bind stray calcium), a gradual decline of the dark current and slowing of responses was usually observed over a period of 10-20 min after rupture of the patch. When the patch pipette contained no added calcium and 10 mM of the calcium chelator BAPTA (1,2-bis(o-aminophenoxy)ethane-N,N,N'',N''-tetraacetic acid) (free Ca2+ ca. 10-9 M) rupture of the patch led, over a period of a few minutes, to (i) an increase in mean dark current, (ii) an increased duration of responses, (iii) a substantial increase in flash sensitivity, and (iv) a pronounced overshoot in the recovery phase, but with (v) virtually no change in the rising phase of the response to bright flashes. Similar results were obtained when EGTA was used in place of BAPTA, and also in the few cases when successful rupture of the outer segment membrane was obtained. With the free calcium concentration in the patch pipette buffered to the higher level of 1 .mu.M (with 10 mM-Ca2+/11 mM-BAPTA) the results were qualitatively similar to those obtained with BAPTA alone, except that the mean dark current did not increase. This is consistent with a resting free calcium concentration in darkness in the region of 1 .mu.M. In the presence of bright steady illumination with BAPTA in the cell the suppression of outer segment current could be maintained for at least 15 min. Upon extinction of the light a very large current transient developed (similar to the overshoot with flashes) which was light suppressible. With backgrounds of moderate intensity the incorporation of buffer led to a gradual reduction of the residual current. From both qualitative and quantitative considerations we interpret the results to be inconsitent with the calcium hypothesis of phototransduction, and to indicate that a rise in cytoplasmic free calcium concentration is not necessary for the light response in rods. Instead the results are consistent with the idea that light normally leads to a reduction in cytoplasmic free calcium concentration.