Control of light-sensitive current in salamander rods.

1. The exponential decline of light-sensitive current seen after switch from Na+ to Li+ in the presence of Ca2+ probably depends on the activity of the phosphodiesterase (PDE) which hydrolyses cyclic GMP. 2. This probability is supported by experiments with suction electrodes which show that in toad and salamander rods the rate constant, b, of the exponential decline of current was increased at least 10-fold by moderate light intensities and decreased about 10-fold by 3-isobutyl-1-methylxanthine (IBMX), an inhibitor of PDE. 3. The rate constant b is about 3 times more sensitive to weak lights or to IBMX than the membrane current. This may be explained by a feed-back involving calcium ions which tends to hold current constant, perhaps by calcium inhibition of guanylate cyclase. 4. The time course of b, which probably represents the changes in PDE activity, was measured by switching from Na+ to Li+ at various times after a flash. The results suggest that a moderate flash (140 Rh) increased b about 7 times in 0.5 s and that b then declined with a time constant of 1.5-2 s. 5. Extrapolated values of the parameter b suggest that strong flashes (5000-10,000 Rh) increased b from 1 s-1 in the dark to perhaps 60 s-1 and that b continued to increase with flash strength for several log units after the current had reached saturation. 6. The observations in 4 and 5 fit well with the idea that b is related to PDE activity and that changes in the latter are sufficient to account for the rising phase of the flash response. 7. After a flash the light-sensitive current recovers much more rapidly than the time constant b-1, a discrepancy which is explained if a light flash causes a delayed increase in guanylate cyclase activity. 8. The apparent delayed increase in cyclase activation is consistent with an inhibitory effect of [Ca2+]i which is reduced when calcium is pumped out during the plateau of the response. 9. Experiments in which pulses of IBMX were applied at different times during a flash response support the idea that a flash causes a delayed increase in the rate of supply of cyclic GMP. Quantitative analysis of these and other tests with IBMX gave rate constants similar to those obtained by the Na+----Li+ method.

Measurement of sodium-calcium exchange in salamander rods.

1. Methods employing suction electrodes to measure the small inward currents associated with the exchange of internal Ca2+ for external Na+ in salamander rod outer segments are described. 2. The ratio of the integral of the exchange current to the integral of the Ca2+ current during the loading period averaged 0.37, which is consistent with 1 Ca2+ ion exchanging with 2.7 Na+ ions, in approximate agreement with Yau & Nakatani (1984b). 3. The transient pumping current observed when external Na+ was restored after a few seconds in isotonic Ca2+ with IBMX (3-isobutyl-1-methylxanthine) consisted of a phase with current at a constant saturated level followed by a phase in which current declined along a characteristic S-shaped curve that was much steeper than expected from the Michaelis equation. 4. The relation between Ca2+ load and pumping current was also steeper than a Michaelis relation. 5. In Ringer solution at 20 degrees C the saturated exchange current was about 20 pA and the value of charge at which the current was half-saturated was 1-5 pC corresponding to 6-30 X 10(6) Ca2+ ions per rod outer segment. 6. The Ca2+ exchange current after small loads declined along the same curve as that determined with medium loads but fell more slowly after large loads. 7. The exchange current at the beginning of the plateau of a strong flash response usually declined along the curve determined with small or medium Ca2+ loads. 8. There was evidence that the exchange current at the tip of the outer segment remained saturated for longer than at the base. 9. The time to pump out Ca2+ through the Na+-Ca2+ exchange system is largely responsible for the delay in the recovery of the light-sensitive current after a Ca2+ load. 10. A theoretical analysis of some of the observations in this and the succeeding paper is based on assumptions about the binding of Ca2+ by exchange sites and by cytoplasmic Ca2+ buffers.

The effect of ions on sodium-calcium exchange in salamander rods.

1. The influence of external cations on the rate at which a Ca2+ load was eliminated in exchange for external Na+ was studied by measuring the inward current associated with Na+-Ca2+ exchange in salamander rods. 2. In Ringer solution the exchange current saturated at a well-defined level of about 20 pA at 20 degrees C. 3. The saturation level of exchange current, j(sat), was increased by lowering the external concentrations of H+, Ca2+, Mg2+ and K+; it was decreased by raising the external concentration of these ions or by lowering [Na+]O. 4. J(sat) varied approximately as [Na+]O2.4 between 35 and 110 mM-Na+. 5. The inhibitory constants for external Ca2+ and Mg2+ were about 1 and 4 mM, respectively. 6. An acid pH decreased j(sat) and an alkaline one increased it; the shape of the relation between current and pH suggests that one inhibitory proton combines between pH 8 and 10 and a pair combine between pH 6 and 7. 7. Removing K+, Mg2+, and Ca2+, and increasing the pH from 7.5 to 10 increased the measured exchange current from 20 to ca. 100 pA. 8. The integral of the Na+-Ca2+ exchange current varied with the Ca2+ load but was largely independent of external ionic changes in spite of large changes in j(sat). The apparent Na+-Ca2+ exchange ratio remained at a little under 3 over a wide range of conditions. 9. The constancy of the integral of the exchange current was brought about by reciprocal variations of the amplitude and duration of the current transient. Records in different solutions could usually be matched by scaling amplitude and time by reciprocal factors. 10. Increasing Nai+ by allowing large light-sensitive currents to flow in low-Ca2+ solutions affected the Na+-Ca2+ exchange transient in a different way from lowering [Na+]o or raising [Ca2+]o, etc. In an Na+-rich rod there was little reduction in j(sat) but the response was prolonged and larger Ca2+ loads were needed to reach saturation. Analysis in terms of a simple model indicated that a substantial Na+ load might reduce the apparent affinity of the internal pumping sites for Ca2+ by a factor of 10. 11. An attempt is made to relate these findings to a model of Na+-Ca2+ exchange.

The ionic selectivity and calcium dependence of the light-sensitive pathway in toad rods.

A new method is described for determining the effects of rapid changes in ionic concentration on the light-sensitive currents of rod outer segments. Replacing Na with another monovalent cation caused a rapid change in current followed by an exponential decline of time constant 0.5-2 s. From the magnitude of the initial rapid change in current we conclude that Li, Na, and K and Rb ions pass readily through the light-sensitive channel in the presence of 1 mM-Ca, whereas Cs crosses with difficulty and choline, tetramethylammonium and tetraethylammonium not at all. The effect of reducing Ca in the external medium indicates that the residual inward current recorded for a few seconds when Na is replaced by an impermeant ion is carried largely by Ca ions. With 1 microM-Ca in the external medium the relative ability of monovalent cations to carry light-sensitive current is Li:Na:K:Rb:Cs = 1.4:1:0.8:0.6:0.15. The same order applied in the physiological region but the values are less certain. Large transient inward currents are seen if external Ca is raised form 1 microM to 5 mM or more; these currents which are maximal in an isotonic Ca solution are presumably carried by Ca. The effect of monovalent cations on the number of open light-sensitive channels was tested by adding the cation to a solution containing 55 mM-Na. Na ions open light-sensitive channels with a delay, probably by promoting Na-Ca exchange; K and Rb close channels by inhibiting exchange; Li and Cs seem inert in the exchange mechanism. The rate at which inward current declines in low [Na]o or high [Ca]o is accelerated by weak background lights and slowed by 3-isobutyl-1-methylxanthine (IBMX), which inhibits the hydrolysis of cGMP. On returning to Ringer solution after a period in low [Na]o the current recovers with a delay of about 1 s which decreases as the Ca concentration of the low [Na]o medium is reduced. We conclude that intracellular Ca has a strong effect on the number of open light-sensitive channels. None the less, several observations are inconsistent with channel closure being dependent simply on combination with internal Ca.

Effect of ions on retinal rods from Bufo marinus.

The effect of ions on the light-sensitive current of isolated retinal rods from the toad Bufo marinus was studied by sucking the inner segment into a tightly fitting pipette. The outer segment projected into flowing solution whose composition could be changed rapidly. Reducing the external Na concentration, [Na]o, round the outer segment caused rapid and reversible reductions in the light-sensitive current. With the outer segment in the pipette, reductions of [Na]o round the inner segment had little effect on the light-sensitive current. The current about 15 s after a change in [Na]o was approximately proportional to [Na]2o. The current decreased in elevated external Ca concentration, [Ca]o, and increased in reduced [Ca]o. Between 10 and 0.5 mM-external Ca the current 15 s after a change was approximately inversely proportional to [Ca]o. Reducing [Ca]o from 1 mM to 1 microM or less transiently increased the current by about 15-fold. After a change in [Na]o or [Ca]o the current did not approach its final value monotonically but with a characteristic overshoot or underswing, followed by a slow relaxation of current which may reflect the time course of change in internal Na. Reducing [Na]o from 110 to 70 mM or less prolonged the response to a flash; very long responses were observed in solutions containing Li rather than Na and also in rods that had been returned to Ringer solution after exposure to low Ca. All these effects might be explained if Ca extrusion in exchange for Na determines the reactivation of current after a flash. The rod current was not changed if the ratio [Na]No/[Ca]o was held constant, N being about 2.5. Between 5 mM and 10 microM-Ca the change in peak current produced by absorption of a single quantum was roughly proportional to the dark current. Responses in the absence of external Na were not normally seen if the solution contained 0.1 mM-Ca or more. Responses of normal polarity were regularly observed in 0 Na, 0 Ca EGTA solutions containing 1.6 mM-Mg. Removal of Mg from such solutions gave inverted responses. Other conditions which promote responses of normal and inverted polarity in Na-free solutions are described briefly. We conclude that Li, Ca, Mg and perhaps K can pass through the light-sensitive channel. The above results suggest that external Na has two distinct effects: (1) it provides ions to carry inward current, and (2) it keeps the light-sensitive conductance open by maintaining the internal Ca concentration, [Ca]i, at a low level.

Effect of ions on the light-sensitive current in retinal rods.

The effect of ions on the light-sensitive current of retinal rods was studied by sucking the inner segment into a tightly fitting capillary with the outer segment projecting into a flowing solution. This new method showed that the light-sensitive pathway, in which Na+ is the normal carrier of current, has an ionic selectivity different from that of other known sodium channels. Externàl calcium has a striking effect on the current, which increased about 20-fold when all calcium was removed. Reducing the sodium concentration gradient greatly prolonged the response to a flash of light, as would be expected if internal calcium blocks sodium channels and if light releases calcium which is subsequently extruded by a sodium-calcium exchange mechanism.

Temporal and spatial characteristics of the voltage response of rods in the retina of the snapping turtle.

1. In response to strong, large-field flashes the dark-adapted rods of Chelydra serpentina gave initial hyperpolarizing responses of 30-40 mV, declining rapidly to plateaus of 10-15 mV which lasted 20 sec or more.2. In the most sensitive cells the flash-sensitivity at 520 nm to a large illuminated area was 3-6 mV per photoisomerization (assuming an effective collecting area of 13.6 mum(2)).3. The initial response to a step of light agreed with that predicted by super-position from the flash response but even with very weak lights the step response fell below the predicted curve at times longer than about 2 sec.4. The step sensitivity defined from the initial peak of the response to a step of light was 2-6 mV photoisomerization(-1) sec, about 1000 times greater than the most sensitive cones in the turtle retina.5. The response to a small weakly illuminated spot (radius 21 mum) reached a peak later and lasted longer than the linear response to a weakly illuminated large area (radius 570 mum).6. The difference in sensitivity between large and small spots was reasonably consistent with the apparent space constant of the rod network obtained from the exponential decline of the flash response on either side of an illuminated strip.7. As others have found, strong flashes did not give an initial hyperpolarizing transient when the radius of the spot was less than about 50 mum.8. Experiments made by flashing long narrow strips of light onto the retina showed that the response spread a long way initially (lambda =... 70 mum) and then contracted down to a relatively small region (lambda =... 25 mum) at times of about 2 sec. When the line source was at some distance from the impaled rod the response reached a peak earlier and was shorter than when the source was close.9. The results in (8) can be explained quantitatively by assuming that delayed voltage-dependent conductance changes mimic an inductance and make the rod network behave like a high-pass filter with series resistance and parallel inductance.10. In sensitive rods, flash responses varied randomly with a variance which was about 1/30 of that expected in an isolated cell; this reduction in noise is satisfactorily explained by electrical coupling between rods.11. The variance peak usually occurred later than the potential peak of the rod response.12. The high-pass filter characteristics of the rod-network help to explain several puzzling features of the behaviour of rods, for example (1), (5), (7), (8) and (11) of this summary.13. The high-pass filter characteristics of the rod-network may help it to optimize the signal to noise ratio by integrating over a large area for rapid signals and over a small one for slow signals.

Electrical coupling between cones in turtle retina.

1. The electrical coupling between cones of known spectral sensitivity in the peripheral part of the turtle's retina was studied by passing current through a micro-electrode inserted into one cone and recording with a second micro-electrode inserted into a neighbouring cone. 2. Spatial sensitivity profiles were determined by recording flash responses to a long narrow strip of light which was moved across the impaled cones in orthogonal directions. These measurements gave both the length constant lambda of electrical spread in the cone network and the separation of the two cones. 3. The cone separation determined from the spatial profiles agreed closely with that measured directly by injecting a fluorescent dye into two cones. 4. The length constant lambda varied from 18 to 39 micron with a mean of 25 micron for red-sensitive cones and 26 micron for green-sensitive cones. 5. The majority of cone pairs studied were electrically coupled provided they had the same spectral sensitivity and were separated by less than 60 micron: thirty-two out of thirty-six red-red pairs, two out of two green-green pairs, none out of eight red-green pairs: no blue cones were observed. 6. The strength of electrical coupling was expressed as a mutual resistance defined as the voltage in one cell divided by the current flowing into the other. Mutual resistances decreased from a maximum value of about 30 M omega at separations close to zero to 0.2 M omega, the lower limit of detectable coupling at separations of about 60 micron. Mutual resistances were always positive and were independent of which cell was directly polarized. The coupling seemed to be ohmic and any rectification or non-linearity probably arose in the cone membranes rather than in the coupling resistances. 7. The results were analysed in terms of the Lamb & Simon (1977) theories of square and hexagonal lattices, which approximate to the continuous sheet model except in the case of the cone to which current is applied. 8. The total membrane resistance of a single cone was estimated as 100--300 M omega and the connecting resistances as 100 M omega for a square array and 170 M omega for a hexagonal array. The input resistance of a cone in the network was 25--50 M omega. Lower values were often obtained but may be due to injury by the micro-electrodes. 9. The time constant of an isolated cone was estimated as about 20 msec and the capacity as about 100 pF. 10. Discrepancies between experimental findings and theoretical predictions of the hexagonal or square array models were tentatively attributed to an overestimate of lambda resulting from light scattering.

A surprising property of electrical spread in the network of rods in the turtle's retina.

Flashing a localised stimulus onto a turtle's retina produces an intracellular potential wave which spreads through electrical connections from illuminated to unilluminated photoreceptors. The response in unilluminated rods (but not in cones) becomes faster as the distance from the source increases, perhaps because voltage-dependent permeability changes in the rod membrane make the coupled network behave like a high-pass filter.

Internal recording of the early receptor potential in turtle cones.

1. Early receptor potentials (E.R.P.s) were recorded with internal electrodes in turtle cones by applying brief flashes from a xenon tube with a maximum photon density equivalent to 2-3 x 10(8) photons micronm-2 at the optimum wave-length. 2. The E.R.P. was separated from the late receptor potential (L.R.P.) by superposing in flash on a step of light which was strong enough to saturate the L.R.P. 3. In red-sensitive cones the E.R.P. consisted of a brief depolarizing phase (R1) followed by a hyperpolarizing phase (R2) of maximum amplitude 10 mV and duration 30-40 msec. R1 was small or absent in green-sensitive cones. 4. With flashes of increasing intensity the E.R.P. approached its maximum exponentially with an exponential constant Q of about 10(8) photons micronm-2 which is of the same order as the reciprocal of the photosensitivity of porphyropsin; the implication of this result, which is considered in the theoretical section, is the the E.R.P. is proportional to the number of photoisomerizations. 5. When tested with a constant xenon flash at varying times after the beginning of a bleaching light the E.R.P. declined exponentially with a similar value of Q. 6. After prolonged bleaches the E.R.P. recovered with a time constant of about 100 sec but much quicker recoveries were observed after relatively brief bleaches. 7. The form and size of the E.R.P. are consistent with the accepted view that it arises from a redistribution of charge in the cone pigment molecule. 8. The effect of a single photoisomerization in an isolated cone was estimated as about 10(-10) V or one electronic charge through about 10% of the membrane.

The electrical response of turtle cones to flashes and steps of light.

1. The linear response of turtle cones to weak flashes or steps of light was usually well fitted by equations based on a chain of six or seven reactions with time constants varying over about a 6-fold range.2. The temperature coefficient (Q(10)) of the reciprocal of the time to peak of the response to a flash was 1.8 (15-25 degrees C), corresponding to an activation energy of 10 kcal/mole.3. Electrical measurements with one internal electrode and a balancing circuit gave the following results on red-sensitive cones of high resistance: resistance across cell surface in dark 50-170 MOmega; time constant in dark 4-6.5 msec. The effect of a bright light was to increase the resistance and time constant by 10-30%.4. If the cell time constant, resting potential and maximum hyperpolarization are known, the fraction of ionic channels blocked by light at any instant can be calculated from the hyperpolarization and its rate of change. At times less than 50 msec the shape of this relation is consistent with the idea that the concentration of a blocking molecule which varies linearly with light intensity is in equilibrium with the fraction of ionic channels blocked.5. The rising phase of the response to flashes and steps of light covering a 10(5)-fold range of intensities is well fitted by a theory in which the essential assumptions are that (i) light starts a linear chain of reactions leading to the production of a substance which blocks ionic channels in the outer segment, (ii) an equilibrium between the blocking molecules and unblocked channels is established rapidly, and (iii) the electrical properties of the cell can be represented by a simple circuit with a time constant in the dark of about 6 msec.6. Deviations from the simple theory which occur after 50 msec are attributed partly to a time-dependent desensitization mechanism and partly to a change in saturation potential resulting from a voltage-dependent change in conductance.7. The existence of several components in the relaxation of the potential to its resting level can be explained by supposing that the ;substance' which blocks light sensitive ionic channels is inactivated in a series of steps.

Changes in time scale and sensitivity in turtle photoreceptors.

1. In turtle cones the steady-state relation between the internal potential and log light intensity was much flatter in the steady state than it was at 30 msec after the beginning of a step of light; this is attributed to a desensitization which develops with a delay of 50-100 msec.2. When a weak flash was superposed on a steady background light which hyperpolarized the cone by 3-6 mV the amplitude of the linear response to a flash was reduced to 1/e and the time to maximum was shortened from about 110 to 70 msec; the response also became diphasic. With stronger background lights the flash sensitivity continued to fall, but the time to maximum did not become shorter than 40-50 msec and lengthened again with very strong lights.3. In cones the flash sensitivity S(F) was reduced to half its dark value S(F) (D) by a light intensity of 1/S(F) (D)zeta where zeta is about 20 sec/V.4. At low levels of background light, about two-thirds of the change in sensitivity was time-dependent and one-third was attributable to the ;instantaneous non-linearity' described in the previous paper.5. The reduction in time to peak and the decrease in sensitivity produced by a background light which hyperpolarized by about 3 mV was little affected by changing the diameter of the area illuminated from 12 to 800 mum.6. An experiment with a rod showed that a very weak light which hyperpolarized by only 0.5 mV decreased the linear response to 1/e and shortened the time to maximum from 300 to 180 msec.7. With weak or moderate flashes the time-dependent desensitization lagged behind the potential by 50-100 msec.8. The desensitization and shortening of time scale which persisted after a flash or step were associated with an after-hyperpolarization. The relaxation of potential, sensitivity and time scale became slower as the preceding illumination was increased from 10(3) to 10(10) photons mum(-2); the increase seemed to occur in steps involving components which relaxed with time constants of the order of 0.1, 1, 10 and 100 sec. A rebound phenomenon was observed after steps longer than 30 sec and with equivalent intensities greater than 10(5) photons mum(-2) sec(-1).9. Several of the observations are explained by a hypothesis in which the central assumption is that the particles which block the ionic channels are degraded or removed by an autocatalytic reaction.

Reconstruction of the electrical responses of turtle cones to flashes and steps of light.

1. Theoretical equations which predict the electrical response of turtle cones to a wide range of light stimuli are developed from the experiments described in previous papers.2. The central points in the theory are that (a) light starts a chain of reactions leading to the production of a substance which blocks ionic channels in the outer segment, (b) an equilibrium between blocking molecules and open channels is rapidly established, (c) the blocking molecules are removed or inactivated by a chain of reactions, the first of which is autocatalytic, (d) in addition to the conductance which decreases with light there is also a conductance which increases with a delay when the cone is hyperpolarized.3. Parameters in the theory were deduced by approximate equations from the experiments described in the previous papers.4. There was good agreement between the properties of real and model cones in the following cases: (a) the response to 10 msec flashes and 0.7 sec steps of light calculated to give between 20 and 5 x 10(7) photoisomerizations per cone at times extending to about 2 sec; (b) the complicated changes in the response to a test flash that occur when it is superposed on background lights of increasing intensity; (c) the after-hyperpolarization and period of reduced sensitivity following a strong flash.5. The main defect of the theory is that the effect of background light in shortening the time to maximum of the response to a flash was more pronounced in a real cone than in the model.