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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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Voltage-induced relief of photocurrent blockade. (A) A photoreceptor stimulated with light (1.5 × 1014 photons s−1 cm−2) was held at −50 mV (where IA is largely inactivated) and Vm was stepped to 0 mV. In normal ASW, the current was biphasic: an abrupt jump, reflecting the increased driving force on potassium, followed by an outward relaxation. After removal of divalents, the relaxation disappeared and the current abruptly attained its steady state amplitude. Traces were normalized. (B) Semi-log plot of the relaxation phase of the current in ASW; the time course was exponential, with a time constant of 8.5 ms.
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Figure 11: Voltage-induced relief of photocurrent blockade. (A) A photoreceptor stimulated with light (1.5 × 1014 photons s−1 cm−2) was held at −50 mV (where IA is largely inactivated) and Vm was stepped to 0 mV. In normal ASW, the current was biphasic: an abrupt jump, reflecting the increased driving force on potassium, followed by an outward relaxation. After removal of divalents, the relaxation disappeared and the current abruptly attained its steady state amplitude. Traces were normalized. (B) Semi-log plot of the relaxation phase of the current in ASW; the time course was exponential, with a time constant of 8.5 ms.

Mentions: To examine the kinetics of unblock of the photocurrent, we used cells screened for a particularly small IA, and restricted the holding potential to −50 mV so that contributions by IA were minimized; unfortunately, at that voltage blockade by divalents is also relatively modest and, therefore, the sensitivity of this test is necessarily reduced. Fig. 11 A shows the results of abruptly depolarizing the membrane to 0 mV during sustained activation of the photocurrent; the procedure was conducted first in normal ASW, and then after removal of Ca2+ and Mg2+. In the presence of divalent cations, the voltage jump produced a rapid step increase in the current, reflecting the increase in driving force on K ions, followed by an outward relaxation. As shown by the semi-logarithmic plot in Fig. 11 B, the relaxation had an exponential time course, with a time constant of 8.5 ms. After removal of Ca2+ and Mg2+, the current became rectangular, directly jumping to the asymptotic amplitude. These observations suggest that the relaxation arises from relief of block by divalent cations (n = 6).


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)

Voltage-induced relief of photocurrent blockade. (A) A photoreceptor stimulated with light (1.5 × 1014 photons s−1 cm−2) was held at −50 mV (where IA is largely inactivated) and Vm was stepped to 0 mV. In normal ASW, the current was biphasic: an abrupt jump, reflecting the increased driving force on potassium, followed by an outward relaxation. After removal of divalents, the relaxation disappeared and the current abruptly attained its steady state amplitude. Traces were normalized. (B) Semi-log plot of the relaxation phase of the current in ASW; the time course was exponential, with a time constant of 8.5 ms.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2230541&req=5

Figure 11: Voltage-induced relief of photocurrent blockade. (A) A photoreceptor stimulated with light (1.5 × 1014 photons s−1 cm−2) was held at −50 mV (where IA is largely inactivated) and Vm was stepped to 0 mV. In normal ASW, the current was biphasic: an abrupt jump, reflecting the increased driving force on potassium, followed by an outward relaxation. After removal of divalents, the relaxation disappeared and the current abruptly attained its steady state amplitude. Traces were normalized. (B) Semi-log plot of the relaxation phase of the current in ASW; the time course was exponential, with a time constant of 8.5 ms.
Mentions: To examine the kinetics of unblock of the photocurrent, we used cells screened for a particularly small IA, and restricted the holding potential to −50 mV so that contributions by IA were minimized; unfortunately, at that voltage blockade by divalents is also relatively modest and, therefore, the sensitivity of this test is necessarily reduced. Fig. 11 A shows the results of abruptly depolarizing the membrane to 0 mV during sustained activation of the photocurrent; the procedure was conducted first in normal ASW, and then after removal of Ca2+ and Mg2+. In the presence of divalent cations, the voltage jump produced a rapid step increase in the current, reflecting the increase in driving force on K ions, followed by an outward relaxation. As shown by the semi-logarithmic plot in Fig. 11 B, the relaxation had an exponential time course, with a time constant of 8.5 ms. After removal of Ca2+ and Mg2+, the current became rectangular, directly jumping to the asymptotic amplitude. These observations suggest that the relaxation arises from relief of block by divalent cations (n = 6).

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