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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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Interaction between calcium blockade and potassium permeation in the light-sensitive conductance. (A) A cell was voltage clamped at −60 mV and stimulated with a 100-ms flash while superfused either with divalent-free solution or after introducing 2 mM Ca2+. (Left) Both extracellular solutions contained the normal potassium concentration (10 mM), whereas (right) the test was performed with elevated [K]o (50 mM), hence the inwardly directed photocurrents. Calcium induced a more pronounced reduction of the light response when it was added to the high-potassium solution. Light intensity 6.1 × 1013 photons s−1 cm−2 (left) and 6.9 × 1014 photons s−1 cm−2 (right). (B) The histogram shows the average blockade for cells tested under the two conditions (n = 3 and 2, respectively).
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Figure 4: Interaction between calcium blockade and potassium permeation in the light-sensitive conductance. (A) A cell was voltage clamped at −60 mV and stimulated with a 100-ms flash while superfused either with divalent-free solution or after introducing 2 mM Ca2+. (Left) Both extracellular solutions contained the normal potassium concentration (10 mM), whereas (right) the test was performed with elevated [K]o (50 mM), hence the inwardly directed photocurrents. Calcium induced a more pronounced reduction of the light response when it was added to the high-potassium solution. Light intensity 6.1 × 1013 photons s−1 cm−2 (left) and 6.9 × 1014 photons s−1 cm−2 (right). (B) The histogram shows the average blockade for cells tested under the two conditions (n = 3 and 2, respectively).

Mentions: To help clarify the site of action of calcium ions, we ascertained possible interactions between blockade and potassium permeation; for example, occlusion of the pore may be alleviated by a knock-off mechanism (Armstrong 1971; Yellen 1984). We addressed this possibility by determining the degree of photocurrent reduction induced by a fixed [Ca2+]o at a steady membrane potential, as the concentration of extracellular potassium was manipulated. Fig. 4 A illustrates a comparison between the responses elicited by a flash of constant intensity at −60 mV, in 0-divalents extracellular solution and after introducing 2 mM Ca2+; the left traces were obtained in the presence of normal [K]o (10 mM), whereas those on the right were recorded with elevated [K]o (50 mM), which made the photocurrent inwardly directed. The extent of blockade was noticeably greater when measured in high-K solution. The histogram in Fig. 4 B shows the average blockade for cells tested under the two conditions (n = 3 and 2). This result suggests a site of block by divalent cations that is located within the permeation pathway of the light-dependent channels.


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)

Interaction between calcium blockade and potassium permeation in the light-sensitive conductance. (A) A cell was voltage clamped at −60 mV and stimulated with a 100-ms flash while superfused either with divalent-free solution or after introducing 2 mM Ca2+. (Left) Both extracellular solutions contained the normal potassium concentration (10 mM), whereas (right) the test was performed with elevated [K]o (50 mM), hence the inwardly directed photocurrents. Calcium induced a more pronounced reduction of the light response when it was added to the high-potassium solution. Light intensity 6.1 × 1013 photons s−1 cm−2 (left) and 6.9 × 1014 photons s−1 cm−2 (right). (B) The histogram shows the average blockade for cells tested under the two conditions (n = 3 and 2, respectively).
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Related In: Results  -  Collection

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Figure 4: Interaction between calcium blockade and potassium permeation in the light-sensitive conductance. (A) A cell was voltage clamped at −60 mV and stimulated with a 100-ms flash while superfused either with divalent-free solution or after introducing 2 mM Ca2+. (Left) Both extracellular solutions contained the normal potassium concentration (10 mM), whereas (right) the test was performed with elevated [K]o (50 mM), hence the inwardly directed photocurrents. Calcium induced a more pronounced reduction of the light response when it was added to the high-potassium solution. Light intensity 6.1 × 1013 photons s−1 cm−2 (left) and 6.9 × 1014 photons s−1 cm−2 (right). (B) The histogram shows the average blockade for cells tested under the two conditions (n = 3 and 2, respectively).
Mentions: To help clarify the site of action of calcium ions, we ascertained possible interactions between blockade and potassium permeation; for example, occlusion of the pore may be alleviated by a knock-off mechanism (Armstrong 1971; Yellen 1984). We addressed this possibility by determining the degree of photocurrent reduction induced by a fixed [Ca2+]o at a steady membrane potential, as the concentration of extracellular potassium was manipulated. Fig. 4 A illustrates a comparison between the responses elicited by a flash of constant intensity at −60 mV, in 0-divalents extracellular solution and after introducing 2 mM Ca2+; the left traces were obtained in the presence of normal [K]o (10 mM), whereas those on the right were recorded with elevated [K]o (50 mM), which made the photocurrent inwardly directed. The extent of blockade was noticeably greater when measured in high-K solution. The histogram in Fig. 4 B shows the average blockade for cells tested under the two conditions (n = 3 and 2). This result suggests a site of block by divalent cations that is located within the permeation pathway of the light-dependent channels.

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