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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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Relaxation of the membrane current after abrupt hyperpolarization. (A) A photoreceptor was voltage clamped at −20 mV and continuously illuminated (1.5 × 1014 photons s−1 cm−2) to activate a steady photocurrent. The voltage was stepped to −60 mV, causing a sudden current jump, due to the reduction in driving force on potassium ions, followed by a slower further decrease in current amplitude. (B) Semi-logarithmic plot of the relaxation phase, showing that its time course followed an exponential time course. (C) Protocol to analyze voltage-induced time-dependent changes in the photocurrent. (Left) A cell was superfused with high-potassium (50 mM) ASW, and the membrane potential was maintained at 0 mV. A sustained step of light was applied (1.5 × 1014 photons s−1 cm−2) and, when the photoresponse was nearly steady, the holding voltage was stepped to −70 mV (L+V). To assess the contribution of non–light-dependent ionic currents, a similar voltage-step was subsequently administered in the dark (V). This current record was subtracted from the previous one to remove leakage, possible voltage-dependent current, and residual uncompensated capacitative currents. (Right) The result is shown, illustrating the time course of the photocurrent alone in response to the voltage jump. The initial inward transient rapidly decays to a reduced plateau level.
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Figure 6: Relaxation of the membrane current after abrupt hyperpolarization. (A) A photoreceptor was voltage clamped at −20 mV and continuously illuminated (1.5 × 1014 photons s−1 cm−2) to activate a steady photocurrent. The voltage was stepped to −60 mV, causing a sudden current jump, due to the reduction in driving force on potassium ions, followed by a slower further decrease in current amplitude. (B) Semi-logarithmic plot of the relaxation phase, showing that its time course followed an exponential time course. (C) Protocol to analyze voltage-induced time-dependent changes in the photocurrent. (Left) A cell was superfused with high-potassium (50 mM) ASW, and the membrane potential was maintained at 0 mV. A sustained step of light was applied (1.5 × 1014 photons s−1 cm−2) and, when the photoresponse was nearly steady, the holding voltage was stepped to −70 mV (L+V). To assess the contribution of non–light-dependent ionic currents, a similar voltage-step was subsequently administered in the dark (V). This current record was subtracted from the previous one to remove leakage, possible voltage-dependent current, and residual uncompensated capacitative currents. (Right) The result is shown, illustrating the time course of the photocurrent alone in response to the voltage jump. The initial inward transient rapidly decays to a reduced plateau level.

Mentions: Our experimental approach to characterizing the kinetics of the interaction between divalent cations and the light-dependent conductance consisted of recording whole-cell currents under voltage clamp and applying perturbations to the command voltage during sustained activation of the light-dependent conductance: an abrupt change in Vm will alter the blockade by divalents, and the resulting reequilibration should manifest itself as a resolvable relaxation, provided its kinetics are sufficiently sluggish. The basic phenomenon is shown in Fig. 6 A: a ciliary photoreceptor was voltage clamped at −20 mV in ASW under continuous illumination and the voltage was stepped to −60 mV, causing an abrupt current jump, as one would expect from the sudden reduction in driving force on K+; additionally, however, a slower further decrease in current is clearly visible. In Fig. 6 B, a semi-logarithmic plot of the tail shows that this relaxation obeyed a single-exponential time course with a time constant of 19 ms.


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)

Relaxation of the membrane current after abrupt hyperpolarization. (A) A photoreceptor was voltage clamped at −20 mV and continuously illuminated (1.5 × 1014 photons s−1 cm−2) to activate a steady photocurrent. The voltage was stepped to −60 mV, causing a sudden current jump, due to the reduction in driving force on potassium ions, followed by a slower further decrease in current amplitude. (B) Semi-logarithmic plot of the relaxation phase, showing that its time course followed an exponential time course. (C) Protocol to analyze voltage-induced time-dependent changes in the photocurrent. (Left) A cell was superfused with high-potassium (50 mM) ASW, and the membrane potential was maintained at 0 mV. A sustained step of light was applied (1.5 × 1014 photons s−1 cm−2) and, when the photoresponse was nearly steady, the holding voltage was stepped to −70 mV (L+V). To assess the contribution of non–light-dependent ionic currents, a similar voltage-step was subsequently administered in the dark (V). This current record was subtracted from the previous one to remove leakage, possible voltage-dependent current, and residual uncompensated capacitative currents. (Right) The result is shown, illustrating the time course of the photocurrent alone in response to the voltage jump. The initial inward transient rapidly decays to a reduced plateau level.
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Related In: Results  -  Collection

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Figure 6: Relaxation of the membrane current after abrupt hyperpolarization. (A) A photoreceptor was voltage clamped at −20 mV and continuously illuminated (1.5 × 1014 photons s−1 cm−2) to activate a steady photocurrent. The voltage was stepped to −60 mV, causing a sudden current jump, due to the reduction in driving force on potassium ions, followed by a slower further decrease in current amplitude. (B) Semi-logarithmic plot of the relaxation phase, showing that its time course followed an exponential time course. (C) Protocol to analyze voltage-induced time-dependent changes in the photocurrent. (Left) A cell was superfused with high-potassium (50 mM) ASW, and the membrane potential was maintained at 0 mV. A sustained step of light was applied (1.5 × 1014 photons s−1 cm−2) and, when the photoresponse was nearly steady, the holding voltage was stepped to −70 mV (L+V). To assess the contribution of non–light-dependent ionic currents, a similar voltage-step was subsequently administered in the dark (V). This current record was subtracted from the previous one to remove leakage, possible voltage-dependent current, and residual uncompensated capacitative currents. (Right) The result is shown, illustrating the time course of the photocurrent alone in response to the voltage jump. The initial inward transient rapidly decays to a reduced plateau level.
Mentions: Our experimental approach to characterizing the kinetics of the interaction between divalent cations and the light-dependent conductance consisted of recording whole-cell currents under voltage clamp and applying perturbations to the command voltage during sustained activation of the light-dependent conductance: an abrupt change in Vm will alter the blockade by divalents, and the resulting reequilibration should manifest itself as a resolvable relaxation, provided its kinetics are sufficiently sluggish. The basic phenomenon is shown in Fig. 6 A: a ciliary photoreceptor was voltage clamped at −20 mV in ASW under continuous illumination and the voltage was stepped to −60 mV, causing an abrupt current jump, as one would expect from the sudden reduction in driving force on K+; additionally, however, a slower further decrease in current is clearly visible. In Fig. 6 B, a semi-logarithmic plot of the tail shows that this relaxation obeyed a single-exponential time course with a time constant of 19 ms.

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