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Divalent cation interactions with light-dependent K channels. Kinetics of voltage-dependent block and requirement for an open pore.

Nasi E, del Pilar Gomez M - J. Gen. Physiol. (1999)

Bottom Line: Both divalents reduce the photocurrent amplitude, the potency being significantly higher for Ca(2+) than Mg(2+) (K(1/2) approximately 16 and 61 mM, respectively, at V(m) = -30 mV).Moreover, conditioning voltage steps terminated immediately before light stimulation failed to affect the photocurrent.Inducing channels to close during a conditioning hyperpolarization resulted in a slight delay in the rising phase of a subsequent light response; this effect can be interpreted as closure of the channel with a divalent ion trapped inside.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology, Boston University School of Medicine, Boston, Massachusetts 02118, USA.

ABSTRACT
The light-dependent K conductance of hyperpolarizing Pecten photoreceptors exhibits a pronounced outward rectification that is eliminated by removal of extracellular divalent cations. The voltage-dependent block by Ca(2+) and Mg(2+) that underlies such nonlinearity was investigated. Both divalents reduce the photocurrent amplitude, the potency being significantly higher for Ca(2+) than Mg(2+) (K(1/2) approximately 16 and 61 mM, respectively, at V(m) = -30 mV). Neither cation is measurably permeant. Manipulating the concentration of permeant K ions affects the blockade, suggesting that the mechanism entails occlusion of the permeation pathway. The voltage dependency of Ca(2+) block is consistent with a single binding site located at an electrical distance of delta approximately 0.6 from the outside. Resolution of light-dependent single-channel currents under physiological conditions indicates that blockade must be slow, which prompted the use of perturbation/relaxation methods to analyze its kinetics. Voltage steps during illumination produce a distinct relaxation in the photocurrent (tau = 5-20 ms) that disappears on removal of Ca(2+) and Mg(2+) and thus reflects enhancement or relief of blockade, depending on the polarity of the stimulus. The equilibration kinetics are significantly faster with Ca(2+) than with Mg(2+), suggesting that the process is dominated by the "on" rate, perhaps because of a step requiring dehydration of the blocking ion to access the binding site. Complementary strategies were adopted to investigate the interaction between blockade and channel gating: the photocurrent decay accelerates with hyperpolarization, but the effect requires extracellular divalents. Moreover, conditioning voltage steps terminated immediately before light stimulation failed to affect the photocurrent. These observations suggest that equilibration of block at different voltages requires an open pore. Inducing channels to close during a conditioning hyperpolarization resulted in a slight delay in the rising phase of a subsequent light response; this effect can be interpreted as closure of the channel with a divalent ion trapped inside.

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Ineffectiveness of voltage perturbations immediately before photostimulation. (A, left) Membrane current measured in a photoreceptor held at 0 mV and stepped for 200 ms to −70 mV. Just after the termination of the voltage pulse, a flash of light was delivered (100 ms, 5.8 × 1014 photons s−1 cm−2). (Center) Effect of the conditioning voltage pulse alone. (Right) Photocurrent in the absence of the prestep. (B) Comparison of the photocurrent elicited with or without a conditioning depolarizing step. The trace with voltage stimulation was corrected for capacitative and leak current by subtracting the record V from L+V. The two photocurrents follow an identical time course. (C) Similar experiment conducted with depolarizing prepulses. In this case, the photoreceptor was voltage clamped at −50 mV and Vm was briefly stepped to +20 mV. (D) Comparison of the leak-corrected photocurrent with or without the conditioning voltage prestep revealed no significant differences in kinetics.
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Figure 13: Ineffectiveness of voltage perturbations immediately before photostimulation. (A, left) Membrane current measured in a photoreceptor held at 0 mV and stepped for 200 ms to −70 mV. Just after the termination of the voltage pulse, a flash of light was delivered (100 ms, 5.8 × 1014 photons s−1 cm−2). (Center) Effect of the conditioning voltage pulse alone. (Right) Photocurrent in the absence of the prestep. (B) Comparison of the photocurrent elicited with or without a conditioning depolarizing step. The trace with voltage stimulation was corrected for capacitative and leak current by subtracting the record V from L+V. The two photocurrents follow an identical time course. (C) Similar experiment conducted with depolarizing prepulses. In this case, the photoreceptor was voltage clamped at −50 mV and Vm was briefly stepped to +20 mV. (D) Comparison of the leak-corrected photocurrent with or without the conditioning voltage prestep revealed no significant differences in kinetics.

Mentions: An alternative approach to testing whether the blockade by divalents interacts with the state of the channel entails applying conditioning voltage steps that are terminated just before the delivery of a light stimulus. If the occupancy of the blocking site by divalents can only change when the channel is in the open conformation (i.e., after photostimulation), then the voltage pre-pulses should have no effect whatsoever. However, if the blocking site is also accessible in the dark (i.e., with the channels closed), then the prepulse should either enhance or depress blockade, depending on the polarity of the stimulus. Such an effect would be expected to linger, owing to the relatively sluggish blocking/unblocking kinetics; as a result, the rising phase of the photocurrent activated immediately after should be affected. Fig. 13 shows the results of an experiment in which a photoreceptor was voltage clamped at a holding potential of 0 mV. The cell was stimulated with a 200-ms voltage step to −70 mV, which terminated immediately before the delivery of a light flash (L+V). A similar voltage step without the flash was also applied (V) to subtract residual leak and capacitative currents. The corrected record was compared with a photocurrent evoked by an identical flash not preceded by the conditioning voltage step (L), as shown in Fig. 13 B: the time course of the two traces is indistinguishable, irrespective of prepulse (n = 5). It should be pointed out that contamination of the rising phase of the light response by the subtraction procedure (owing to possible light-induced changes of IA time course) is negligible here for two reasons. (a) The prestep voltage was chosen to lie near the midpoint of the h∞ curve and its short duration (although >>τ of blockade equilibration) only allows a fraction of the recovery from inactivation that can be attained at that Vm (≈60% of the asymptotic level; data not shown). As a result, IA is reduced by ≈65%. (b) The brief flash followed the voltage transition, precluding the development of any significant modulatory effect on the kinetics of IA.


Divalent cation interactions with light-dependent K channels. Kinetics of voltage-dependent block and requirement for an open pore.

Nasi E, del Pilar Gomez M - J. Gen. Physiol. (1999)

Ineffectiveness of voltage perturbations immediately before photostimulation. (A, left) Membrane current measured in a photoreceptor held at 0 mV and stepped for 200 ms to −70 mV. Just after the termination of the voltage pulse, a flash of light was delivered (100 ms, 5.8 × 1014 photons s−1 cm−2). (Center) Effect of the conditioning voltage pulse alone. (Right) Photocurrent in the absence of the prestep. (B) Comparison of the photocurrent elicited with or without a conditioning depolarizing step. The trace with voltage stimulation was corrected for capacitative and leak current by subtracting the record V from L+V. The two photocurrents follow an identical time course. (C) Similar experiment conducted with depolarizing prepulses. In this case, the photoreceptor was voltage clamped at −50 mV and Vm was briefly stepped to +20 mV. (D) Comparison of the leak-corrected photocurrent with or without the conditioning voltage prestep revealed no significant differences in kinetics.
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Related In: Results  -  Collection

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Figure 13: Ineffectiveness of voltage perturbations immediately before photostimulation. (A, left) Membrane current measured in a photoreceptor held at 0 mV and stepped for 200 ms to −70 mV. Just after the termination of the voltage pulse, a flash of light was delivered (100 ms, 5.8 × 1014 photons s−1 cm−2). (Center) Effect of the conditioning voltage pulse alone. (Right) Photocurrent in the absence of the prestep. (B) Comparison of the photocurrent elicited with or without a conditioning depolarizing step. The trace with voltage stimulation was corrected for capacitative and leak current by subtracting the record V from L+V. The two photocurrents follow an identical time course. (C) Similar experiment conducted with depolarizing prepulses. In this case, the photoreceptor was voltage clamped at −50 mV and Vm was briefly stepped to +20 mV. (D) Comparison of the leak-corrected photocurrent with or without the conditioning voltage prestep revealed no significant differences in kinetics.
Mentions: An alternative approach to testing whether the blockade by divalents interacts with the state of the channel entails applying conditioning voltage steps that are terminated just before the delivery of a light stimulus. If the occupancy of the blocking site by divalents can only change when the channel is in the open conformation (i.e., after photostimulation), then the voltage pre-pulses should have no effect whatsoever. However, if the blocking site is also accessible in the dark (i.e., with the channels closed), then the prepulse should either enhance or depress blockade, depending on the polarity of the stimulus. Such an effect would be expected to linger, owing to the relatively sluggish blocking/unblocking kinetics; as a result, the rising phase of the photocurrent activated immediately after should be affected. Fig. 13 shows the results of an experiment in which a photoreceptor was voltage clamped at a holding potential of 0 mV. The cell was stimulated with a 200-ms voltage step to −70 mV, which terminated immediately before the delivery of a light flash (L+V). A similar voltage step without the flash was also applied (V) to subtract residual leak and capacitative currents. The corrected record was compared with a photocurrent evoked by an identical flash not preceded by the conditioning voltage step (L), as shown in Fig. 13 B: the time course of the two traces is indistinguishable, irrespective of prepulse (n = 5). It should be pointed out that contamination of the rising phase of the light response by the subtraction procedure (owing to possible light-induced changes of IA time course) is negligible here for two reasons. (a) The prestep voltage was chosen to lie near the midpoint of the h∞ curve and its short duration (although >>τ of blockade equilibration) only allows a fraction of the recovery from inactivation that can be attained at that Vm (≈60% of the asymptotic level; data not shown). As a result, IA is reduced by ≈65%. (b) The brief flash followed the voltage transition, precluding the development of any significant modulatory effect on the kinetics of IA.

Bottom Line: Both divalents reduce the photocurrent amplitude, the potency being significantly higher for Ca(2+) than Mg(2+) (K(1/2) approximately 16 and 61 mM, respectively, at V(m) = -30 mV).Moreover, conditioning voltage steps terminated immediately before light stimulation failed to affect the photocurrent.Inducing channels to close during a conditioning hyperpolarization resulted in a slight delay in the rising phase of a subsequent light response; this effect can be interpreted as closure of the channel with a divalent ion trapped inside.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology, Boston University School of Medicine, Boston, Massachusetts 02118, USA.

ABSTRACT
The light-dependent K conductance of hyperpolarizing Pecten photoreceptors exhibits a pronounced outward rectification that is eliminated by removal of extracellular divalent cations. The voltage-dependent block by Ca(2+) and Mg(2+) that underlies such nonlinearity was investigated. Both divalents reduce the photocurrent amplitude, the potency being significantly higher for Ca(2+) than Mg(2+) (K(1/2) approximately 16 and 61 mM, respectively, at V(m) = -30 mV). Neither cation is measurably permeant. Manipulating the concentration of permeant K ions affects the blockade, suggesting that the mechanism entails occlusion of the permeation pathway. The voltage dependency of Ca(2+) block is consistent with a single binding site located at an electrical distance of delta approximately 0.6 from the outside. Resolution of light-dependent single-channel currents under physiological conditions indicates that blockade must be slow, which prompted the use of perturbation/relaxation methods to analyze its kinetics. Voltage steps during illumination produce a distinct relaxation in the photocurrent (tau = 5-20 ms) that disappears on removal of Ca(2+) and Mg(2+) and thus reflects enhancement or relief of blockade, depending on the polarity of the stimulus. The equilibration kinetics are significantly faster with Ca(2+) than with Mg(2+), suggesting that the process is dominated by the "on" rate, perhaps because of a step requiring dehydration of the blocking ion to access the binding site. Complementary strategies were adopted to investigate the interaction between blockade and channel gating: the photocurrent decay accelerates with hyperpolarization, but the effect requires extracellular divalents. Moreover, conditioning voltage steps terminated immediately before light stimulation failed to affect the photocurrent. These observations suggest that equilibration of block at different voltages requires an open pore. Inducing channels to close during a conditioning hyperpolarization resulted in a slight delay in the rising phase of a subsequent light response; this effect can be interpreted as closure of the channel with a divalent ion trapped inside.

Show MeSH
Related in: MedlinePlus