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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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Removal of divalent cations eliminates the photocurrent relaxations induced by voltage perturbations. (A) The effects of voltage steps on the current through the light-sensitive channels was compared in the presence (left) and absence (right) of extracellular Ca2+ and Mg2+. After removal of divalent cations, a hyperpolarizing step from 0 to −70 mV elicited a downward shift in membrane current, of comparable amplitude as the control trace, but lacking the rapid relaxation. (B) Normalized, superimposed recordings shown in an expanded time scale, to underscore the disappearance of the relaxation in divalent-free solution. All the current records were obtained in the presence of 50 mM extracellular potassium and were corrected for capacitative and non-light-dependent ionic currents. Light intensity 1.5 × 1014 photons s−1 cm−2.
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Figure 7: Removal of divalent cations eliminates the photocurrent relaxations induced by voltage perturbations. (A) The effects of voltage steps on the current through the light-sensitive channels was compared in the presence (left) and absence (right) of extracellular Ca2+ and Mg2+. After removal of divalent cations, a hyperpolarizing step from 0 to −70 mV elicited a downward shift in membrane current, of comparable amplitude as the control trace, but lacking the rapid relaxation. (B) Normalized, superimposed recordings shown in an expanded time scale, to underscore the disappearance of the relaxation in divalent-free solution. All the current records were obtained in the presence of 50 mM extracellular potassium and were corrected for capacitative and non-light-dependent ionic currents. Light intensity 1.5 × 1014 photons s−1 cm−2.

Mentions: The next step was to determine the relationship between these relaxations and blockade by divalents. To this end, the effects of voltage perturbations applied in the presence vs. absence of extracellular Ca2+ and Mg2+ were compared. Fig. 7 A demonstrates that after removal of divalent cations, a hyperpolarizing step from 0 to −70 mV elicited a downward shift in membrane current that totally lacked the rapid relaxation. The different time course obtained in the two cases is highlighted in Fig. 7 B, which shows the two normalized superimposed traces in an expanded time scale (n = 5).


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)

Removal of divalent cations eliminates the photocurrent relaxations induced by voltage perturbations. (A) The effects of voltage steps on the current through the light-sensitive channels was compared in the presence (left) and absence (right) of extracellular Ca2+ and Mg2+. After removal of divalent cations, a hyperpolarizing step from 0 to −70 mV elicited a downward shift in membrane current, of comparable amplitude as the control trace, but lacking the rapid relaxation. (B) Normalized, superimposed recordings shown in an expanded time scale, to underscore the disappearance of the relaxation in divalent-free solution. All the current records were obtained in the presence of 50 mM extracellular potassium and were corrected for capacitative and non-light-dependent ionic currents. Light intensity 1.5 × 1014 photons s−1 cm−2.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 7: Removal of divalent cations eliminates the photocurrent relaxations induced by voltage perturbations. (A) The effects of voltage steps on the current through the light-sensitive channels was compared in the presence (left) and absence (right) of extracellular Ca2+ and Mg2+. After removal of divalent cations, a hyperpolarizing step from 0 to −70 mV elicited a downward shift in membrane current, of comparable amplitude as the control trace, but lacking the rapid relaxation. (B) Normalized, superimposed recordings shown in an expanded time scale, to underscore the disappearance of the relaxation in divalent-free solution. All the current records were obtained in the presence of 50 mM extracellular potassium and were corrected for capacitative and non-light-dependent ionic currents. Light intensity 1.5 × 1014 photons s−1 cm−2.
Mentions: The next step was to determine the relationship between these relaxations and blockade by divalents. To this end, the effects of voltage perturbations applied in the presence vs. absence of extracellular Ca2+ and Mg2+ were compared. Fig. 7 A demonstrates that after removal of divalent cations, a hyperpolarizing step from 0 to −70 mV elicited a downward shift in membrane current that totally lacked the rapid relaxation. The different time course obtained in the two cases is highlighted in Fig. 7 B, which shows the two normalized superimposed traces in an expanded time scale (n = 5).

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