Ionic movements through light‐sensitive channels of toad rods.

160

151

1. Membrane current was recorded from an isolated, dark‐adapted toad rod by sucking either its inner segment or outer segment into a tight‐fitting glass pipette containing Ringer solution. The remainder of the cell was exposed to bath solution which could be changed rapidly. 2. In normal Ringer solution the current response of a cell to a saturating flash or step of light showed a small secondary rise at its initial peak. The profile of this secondary rise (i.e. amplitude and time course) was independent of both the intensity and the duration of illumination once the light response had reached a plateau level. 3. This secondary rise disappeared when external Na+ around the outer segment was replaced by Li+ or guanidinium, suggesting that it represented an electrogenic Na+‐dependent Ca2+ efflux which was declining after the onset of light. 4. This Na+‐Ca2+ exchange activity showed a roughly exponential decline, with a time constant of about 0.5 s. Exponential extrapolation of the exchange current to the time at half‐height of the light response gave an initial amplitude of about 2 pA. Using La3+ as a blocker, we did not detect any steady exchange current after the initial exponential decline. 5. An intense flash superposed on a just‐saturating steady background light failed to produce any incremental exchange current transient. 6. Our interpretation of the above results is that in darkness there are counterbalancing levels of Ca2+ influx (through the light‐sensitive conductance) and efflux (through the Na+‐Ca2+ exchange) across the plasma membrane of the rod outer segment. The exchange current transient at the onset of light merely represents the unidirectional Ca2+ efflux which becomes revealed as a result of the stoppage of the Ca2+ influx, rather than a de novo Ca2+ efflux triggered by light. 7. Consistent with this interpretation, a test light delivered soon after a saturating, conditioning light elicited little exchange current, which then gradually recovered to control value with a time course parallel to the restoration of the dark current. Conversely, when the dark current was increased above its physiological level by IBMX (isobutylmethylxanthine) the exchange current transient became larger than control.(ABSTRACT TRUNCATED AT 400 WORDS)

Section: Composition Of the Dark Currentmentioning

confidence: 81%

Calcium and magnesium fluxes across the plasma membrane of the toad rod outer segment.

Nakatani

Yau

1988

160

151

“…Alternatively it is possible that lowered calcium functions by decreasing the activity of the phosphodiesterase (Bownds, 1980), either directly or through modification of earlier biochemical steps in the transduction pathway (Lamb et al 1985b). Whatever the mechanism, one result of an elevated dark current would be an elevated influx of Ca2+, because the light-sensitive channels are permeable to Ca2+ 345 T. D. LAMB, H. R. MATTHEWS AND V. TORRE and other divalent ions (Capovilla, Caretta, Cervetto & Torre, 1983;Hodgkin et al 1984;Yau & Nakatani, 1984a, b). This would tend to oppose the reduction in cytoplasmic free calcium concentration, thus completing the negative feed-back loop.…”

Section: Cytoplasmic Free Calcium Concentrationmentioning

confidence: 99%

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

Lamb¹,

Matthews²,

Torre³

1986

Self Cite

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.

“…The selectivity of the cGMP-activated channel to alkali monovalent cations and to divalent cations has already been studied both in intact retinal rods, by changing solutions at the extracellular side of the cell (Capovilla, Caretta, Cervetto & Torre, 1983;Hodgkin, McNaughton & Nunn, 1985;Nakatani & Yau, 1988;Menini, Rispoli & Torre, 1988) and in excised inside-out patches by changing internal cations (Fesenko et al 1985;Nunn, 1987;Furman & Tanaka, 1990;Menini 1990;Colamartino, Menini & Torre, 1991). It was found that the cGMP-activated channel is a poorly selective cation channel with different selectivity properties from those of other channels.…”

Section: Introductionmentioning

confidence: 99%

The permeability of the cGMP‐activated channel to organic cations in retinal rods of the tiger salamander.

Picco

Menini

1993

SUMMARY1. The permeability of the channel activated by guanosine 3',5'-cyclic monophosphate (cGMP) to many organic monovalent cations was determined by recording macroscopic currents in excised inside-out patches of plasma membrane from isolated retinal rod outer segments of the tiger salamander.2. Current-voltage relations were measured when the NaCl of the bathing medium was replaced by salts of organic cations. Permeability ratios relative to Na+ ions were calculated with the Goldman-Hodgkin-Katz potential equation from the measured changes of reversal potentials.3. Hydroxylammonium+, hydrazinium+ and methylammonium+, which are molecules of very similar shape and size, permeate the channel with very different permeability ratios: 5-92, 1-99 and 0 60 respectively. 4. Methylated and ethylated ammonium+ compounds were investigated. It was found that, not only methylammonium+, but also dimethylammonium+ and ethylammonium+ were permeant with permeability ratios of 0-6, 0 14 and 0 16 respectively. Trimethylammonium+, tetramethylammonium+, diethylammonium+, triethylammonium+ and tetraethylammonium+ were not permeant.5. Guanidinium+ and its derivatives formamidinium+, aminoguanidinium+, acetamidinium+ and methylguanidinium+ were all permeant with permeability ratios 1-12, 1.00, 0-63, 0-36 and 0 33 respectively. 6. The cGMP-activated channel was found to be permeable to at least thirteen organic cations. Molecular models of the permeant cations indicate that the crosssection of the narrowest part of the pore must be at least as large as a rectangle of 0-38 x 0 5 nm dimensions.