The effects of sodium replacement on the responses of toad rods

129

SUMMARY1. 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.2. 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.3. 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.4. 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.5. 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.6. With the free calcium concentration in the patch pipette buffered to the higher level of 1 JuM (with 10 mm-Ca2+/1 1 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 fUM.7. 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 T. D. LAMB, H. R. MATTHEWS AND V. TORRE intensity the incorporation of buffer led to a gradual reduction of the residual current.8. From both qualitative and quantitative considerations we interpret the results to be inconsistent 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.

Section: Patch-pipette Solutionsmentioning

confidence: 54%

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

Lamb¹,

Matthews²,

Torre³

1986

129

“…Recently, however, Fain & Lisman (1981) and Bastian & Fain (1982) reported that when external Ca2+ is buffered to about 10-8 M, voltage responses can be recorded when Na+ is substituted for Li+. Current responses to light have been recorded when Na+ was completely replaced by choline in low Ca2+ solutions (Yau et al 1981).…”

Section: Capo Villa and Othersmentioning

confidence: 99%

Ionic movements through light‐sensitive channels of toad rods.

Capovilla¹,

Caretta²,

Cervetto³

et al. 1983

SUMMARY1. Electrical photoresponses of rods in the isolated toad retina were recorded during ionic manipulations of the Na+-free extracellular medium.2. In the presence of a concentration of external Ca2+ above 10-5 M, voltage photoresponses were observed only in the presence of external Na+ or Li+.3. When external Ca2+ was reduced below 10-6 M, voltage photoresponses of normal polarity could be detected even in the absence of Na+ or Li+, but in the presence of external Mg2+.4. In the presence of normal extracellular Ca2+ hyperpolarizing photoresponses were observed even in the absence of Na+ or Li+, provided small amounts of phosphodiesterase inhibitors (IBMX, RO 20-1724, papaverine, caffeine, theophylline) were added to the perfusate.5. Responses obtained in low-Na+ IBMX solutions required the presence of millimolar amounts of a variety ofdivalent cations, among which Mn2+ and Ba2+ were the most effective.6. When the concentration of both external Ca2+ and Mg2+ was reduced to micromolar amounts, depolarizing photoresponses were observed. In these conditions measurements with radioactive tracers showed a light-modulated efflux of 42K+ or 86Rb+.7. The light-modulated 42K+ or 86Rb+ efflux was halved by 2-6 mM-external K+ and was completely blocked when K+ was raised above 10 mM.8. These results show that ionic movements through light-sensitive channels are controlled by Ca2+ and Mg2+ and possibly also be the intracellular level of cyclic nucleotides. Moreover, the movement of ions through the light-sensitive channel, does not obey the independence principle.

“…Evidence that this is indeed the case comes from studies in which Li+ is substituted for Na+ in the perfusate. When the superfusate [Ca2+1] is less than or equal to 10-7 M, Li+ may be substituted for Na+ without eliminating the photoresponse (solutions L to 0) (Fain & Lisman, 1981;Yau et al 1981;Bastian & Fain, 1982b (Bastian & Fain, 1982b; but see Yau et al 1981).…”

mentioning

confidence: 99%

Adapting lights and lowered extracellular free calcium desensitize toad photoreceptors by differing mechanisms.

Greenblatt¹

1983

SUMMARY1. Extracellular recordings were made across the outer segment layer of isolated, superfused toad retinas. Under these recording conditions, the photovoltage reflects primarily the current flowing through the outer-segment membrane of red rods.2. In normal toad Ringer solution, a dim conditioning flash desensitized a test flash response. The desensitization reached a peak 1-8-2-0 s after the conditioning flash and then declined approximately as an exponential with time constant 6 s. 6. These results can be understood in terms of the Ca2+ hypothesis of transduction (Hagins & Yoshikami, 1974) if it is assumed that lowered [Ca2+]0 exposes an endogenous Ca2+ buffer. The data also provide evidence for a role of Na+/Ca2+ exchange in regulating intracellular Ca2+ concentration in the toad photoreceptor.7. A quantitative model based on these assumptions is derived and compared with the experimental data.