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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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Concentration dependence of the photocurrent blockade by extracellular Ca2+. (A) Normalized peak amplitude of the light response measured at −30 mV in different photoreceptors. After four flashes, the solution was switched from 0-divalents to the indicated concentration of calcium. (B) Average blockade of the photocurrent obtained from 12 cells, each tested in 0-divalents and in one concentration of Ca2+. A Langmuir function was fitted to the data point by the method of least squares; the half-maximal blockade was attained at 16 mM Ca2+. (C) Graded outward rectification induced by two different Ca2+ concentrations in the same cell. Photocurrents were recorded at potentials varying from −60 to +20 mV in 10-mV increments in each of the solutions indicated. (D) plots for the data shown in C, illustrating the progressively greater outward rectification as Ca2+ concentration was increased. Light intensity: 2.4 × 1014 photons s−1 cm−2.
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Figure 3: Concentration dependence of the photocurrent blockade by extracellular Ca2+. (A) Normalized peak amplitude of the light response measured at −30 mV in different photoreceptors. After four flashes, the solution was switched from 0-divalents to the indicated concentration of calcium. (B) Average blockade of the photocurrent obtained from 12 cells, each tested in 0-divalents and in one concentration of Ca2+. A Langmuir function was fitted to the data point by the method of least squares; the half-maximal blockade was attained at 16 mM Ca2+. (C) Graded outward rectification induced by two different Ca2+ concentrations in the same cell. Photocurrents were recorded at potentials varying from −60 to +20 mV in 10-mV increments in each of the solutions indicated. (D) plots for the data shown in C, illustrating the progressively greater outward rectification as Ca2+ concentration was increased. Light intensity: 2.4 × 1014 photons s−1 cm−2.

Mentions: The apparent affinity of Ca2+for the channel was determined as illustrated in Fig. 3. Photoreceptors were voltage clamped at −30 mV and stimulated with repetitive flashes of constant intensity, initially in the absence of all divalents, and then after introducing either 2, 10, 30, or 60 mM Ca2+ (0-Mg). For comparison purposes, responses were normalized with respect to the maximum amplitude attained during the control trials in 0-divalents. Calcium depressed the light-evoked current in a concentration-dependent way. To quantify the dose dependency of the blockade, data from 12 cells tested with the same light intensity were pooled and plotted in Fig. 3 B. The smooth curve represents a Langmuir function fitted to the average data points by the method of least squares; half-maximal block was attained at ≈16 mM. A Hill function provided no better fit, and the resulting Hill coefficient was not significantly different from 1 (0.91), suggesting that a single calcium ion blocks the channel. Similar measurements were conducted at 0 mV, and the obtained K1/2 was ≈61 mM for calcium. The corresponding estimate for Mg2+ would be difficult to obtain because of its lower affinity: 60 mM Mg2+ only reduced the photocurrent by 12.5% (n = 2), so that an extrapolated figure would be >>100 mM. The remaining parts of Fig. 3 illustrate the concentration-dependent induction of outward rectification in the photocurrent by extracellular Ca2+. In Fig. 3 C, families of light-evoked currents were measured as the holding potential was stepped from −60 to +20 mV in 10-mV increments; the experiment was conducted initially in divalent-free solution, and subsequently repeated in the presence of 10 and 60 mM Ca2+ (in the same cell). As [Ca2+]o was raised, the size of the currents evoked at the more negative voltages became compressed. The peak amplitude of the responses, plotted in D, clearly illustrates the progressively increasing curvature in the I–V relation. Comparable dose-dependent effects were observed in another photoreceptor tested with 0, 2, and 10 mM Ca2+, and 10 additional cells in which the 0-divalent solution was compared with one fixed calcium concentration, in the range of 2–60 mM.


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)

Concentration dependence of the photocurrent blockade by extracellular Ca2+. (A) Normalized peak amplitude of the light response measured at −30 mV in different photoreceptors. After four flashes, the solution was switched from 0-divalents to the indicated concentration of calcium. (B) Average blockade of the photocurrent obtained from 12 cells, each tested in 0-divalents and in one concentration of Ca2+. A Langmuir function was fitted to the data point by the method of least squares; the half-maximal blockade was attained at 16 mM Ca2+. (C) Graded outward rectification induced by two different Ca2+ concentrations in the same cell. Photocurrents were recorded at potentials varying from −60 to +20 mV in 10-mV increments in each of the solutions indicated. (D) plots for the data shown in C, illustrating the progressively greater outward rectification as Ca2+ concentration was increased. Light intensity: 2.4 × 1014 photons s−1 cm−2.
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Figure 3: Concentration dependence of the photocurrent blockade by extracellular Ca2+. (A) Normalized peak amplitude of the light response measured at −30 mV in different photoreceptors. After four flashes, the solution was switched from 0-divalents to the indicated concentration of calcium. (B) Average blockade of the photocurrent obtained from 12 cells, each tested in 0-divalents and in one concentration of Ca2+. A Langmuir function was fitted to the data point by the method of least squares; the half-maximal blockade was attained at 16 mM Ca2+. (C) Graded outward rectification induced by two different Ca2+ concentrations in the same cell. Photocurrents were recorded at potentials varying from −60 to +20 mV in 10-mV increments in each of the solutions indicated. (D) plots for the data shown in C, illustrating the progressively greater outward rectification as Ca2+ concentration was increased. Light intensity: 2.4 × 1014 photons s−1 cm−2.
Mentions: The apparent affinity of Ca2+for the channel was determined as illustrated in Fig. 3. Photoreceptors were voltage clamped at −30 mV and stimulated with repetitive flashes of constant intensity, initially in the absence of all divalents, and then after introducing either 2, 10, 30, or 60 mM Ca2+ (0-Mg). For comparison purposes, responses were normalized with respect to the maximum amplitude attained during the control trials in 0-divalents. Calcium depressed the light-evoked current in a concentration-dependent way. To quantify the dose dependency of the blockade, data from 12 cells tested with the same light intensity were pooled and plotted in Fig. 3 B. The smooth curve represents a Langmuir function fitted to the average data points by the method of least squares; half-maximal block was attained at ≈16 mM. A Hill function provided no better fit, and the resulting Hill coefficient was not significantly different from 1 (0.91), suggesting that a single calcium ion blocks the channel. Similar measurements were conducted at 0 mV, and the obtained K1/2 was ≈61 mM for calcium. The corresponding estimate for Mg2+ would be difficult to obtain because of its lower affinity: 60 mM Mg2+ only reduced the photocurrent by 12.5% (n = 2), so that an extrapolated figure would be >>100 mM. The remaining parts of Fig. 3 illustrate the concentration-dependent induction of outward rectification in the photocurrent by extracellular Ca2+. In Fig. 3 C, families of light-evoked currents were measured as the holding potential was stepped from −60 to +20 mV in 10-mV increments; the experiment was conducted initially in divalent-free solution, and subsequently repeated in the presence of 10 and 60 mM Ca2+ (in the same cell). As [Ca2+]o was raised, the size of the currents evoked at the more negative voltages became compressed. The peak amplitude of the responses, plotted in D, clearly illustrates the progressively increasing curvature in the I–V relation. Comparable dose-dependent effects were observed in another photoreceptor tested with 0, 2, and 10 mM Ca2+, and 10 additional cells in which the 0-divalent solution was compared with one fixed calcium concentration, in the range of 2–60 mM.

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