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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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Comparison of the effects of Ca2+ and Mg2+ on photocurrent changes after hyperpolarizing steps. (A) Superimposed normalized currents evoked by steps of voltage from 0 to −70 mV in 0-divalents, 60 mM Ca2+ or 60 mM Mg2; in each case a 2-s light (2.4 × 1014 photons s−1 cm−2) was turned on 1.3 s before the change in Vm. (B) Effect of varying the size of the voltage step. A different photoreceptor was stimulated with a similar protocol, except that the voltage was stepped to −50, −60, −70, and −80 mV on succeeding trials. In divalent-free solution, the current change after the voltage step had a nearly rectangular time course and an amplitude proportional to the voltage stimulus. In the presence of Ca2+, the current swiftly decayed to a small-amplitude plateau level that was graded with Vm. A qualitatively similar effect occurred with Mg2+, but the effect was much reduced, both in terms of relaxation amplitude and speed. The dotted lines mark the 0-current level. (C) Plot of the time constant obtained by fitting single exponential functions to the relaxations measured in 60 mM Ca2+, in response to steps to different voltages from a holding potential of 0 mV. Each point is the average of three cells; error bars represent standard deviations.
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Figure 8: Comparison of the effects of Ca2+ and Mg2+ on photocurrent changes after hyperpolarizing steps. (A) Superimposed normalized currents evoked by steps of voltage from 0 to −70 mV in 0-divalents, 60 mM Ca2+ or 60 mM Mg2; in each case a 2-s light (2.4 × 1014 photons s−1 cm−2) was turned on 1.3 s before the change in Vm. (B) Effect of varying the size of the voltage step. A different photoreceptor was stimulated with a similar protocol, except that the voltage was stepped to −50, −60, −70, and −80 mV on succeeding trials. In divalent-free solution, the current change after the voltage step had a nearly rectangular time course and an amplitude proportional to the voltage stimulus. In the presence of Ca2+, the current swiftly decayed to a small-amplitude plateau level that was graded with Vm. A qualitatively similar effect occurred with Mg2+, but the effect was much reduced, both in terms of relaxation amplitude and speed. The dotted lines mark the 0-current level. (C) Plot of the time constant obtained by fitting single exponential functions to the relaxations measured in 60 mM Ca2+, in response to steps to different voltages from a holding potential of 0 mV. Each point is the average of three cells; error bars represent standard deviations.

Mentions: Considering the disparate potency of voltage-dependent block by Ca2+ and Mg2+ at steady holding potentials (Fig. 2), some mechanistic insight can be gained by comparing their respective kinetics by perturbation/relaxation analysis. Fig. 8 A shows the normalized currents evoked by a hyperpolarizing step from 0 to −70 mV during illumination, in a cell that was successively superfused with extracellular solution containing elevated K (50 mM), either devoid of divalents or in the presence of 60 mM Ca2+ or 60 mM Mg2+. As before, in 0-divalents, the photocurrent remained stationary after the voltage perturbation, whereas a conspicuous relaxation occurred in the presence of either divalent cation; the most striking difference, however, is that the time course with calcium (τ = 7 ms) was much faster than with magnesium (τ = 29 ms). In both ionic conditions, the relaxations accelerated as a function of the membrane hyperpolarization: in Fig. 8 B, the voltage was stepped in 10-mV increments between −50 and −80 mV, from a holding potential of 0 mV. It is clear that both the amplitude and the speed of the relaxation are graded with the size of the voltage stimulus, although with Mg2+ these transients remained significantly smaller and slower. By contrast, in 0-divalents, the currents after each step retained a nearly flat time course (except for some slow creep), and their amplitude changed linearly with voltage. These effects were confirmed in a total of five cells (two tested with a shortened protocol). The range of voltages examined could not be extended further, because the applied stimulus had to be significantly more negative than approximately −40 mV to insure a reasonable driving force, but not too large, otherwise membrane breakdown occurs (Gomez and Nasi 1994a), a situation that is exacerbated by removal of divalents (notice the noisy traces in Fig. 1 A and 7 B). The progressive shortening of the relaxation time constant with membrane hyperpolarization in the presence of Ca2+ is illustrated in Fig. 8 C; each point in the plot represents an average value (n = 3). The corresponding data for magnesium (not shown) were more complex in that the sum of two exponential functions was often required to satisfactorily fit the data, with the relative contribution of the two components changing significantly as a function of voltage.


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)

Comparison of the effects of Ca2+ and Mg2+ on photocurrent changes after hyperpolarizing steps. (A) Superimposed normalized currents evoked by steps of voltage from 0 to −70 mV in 0-divalents, 60 mM Ca2+ or 60 mM Mg2; in each case a 2-s light (2.4 × 1014 photons s−1 cm−2) was turned on 1.3 s before the change in Vm. (B) Effect of varying the size of the voltage step. A different photoreceptor was stimulated with a similar protocol, except that the voltage was stepped to −50, −60, −70, and −80 mV on succeeding trials. In divalent-free solution, the current change after the voltage step had a nearly rectangular time course and an amplitude proportional to the voltage stimulus. In the presence of Ca2+, the current swiftly decayed to a small-amplitude plateau level that was graded with Vm. A qualitatively similar effect occurred with Mg2+, but the effect was much reduced, both in terms of relaxation amplitude and speed. The dotted lines mark the 0-current level. (C) Plot of the time constant obtained by fitting single exponential functions to the relaxations measured in 60 mM Ca2+, in response to steps to different voltages from a holding potential of 0 mV. Each point is the average of three cells; error bars represent standard deviations.
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Related In: Results  -  Collection

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Figure 8: Comparison of the effects of Ca2+ and Mg2+ on photocurrent changes after hyperpolarizing steps. (A) Superimposed normalized currents evoked by steps of voltage from 0 to −70 mV in 0-divalents, 60 mM Ca2+ or 60 mM Mg2; in each case a 2-s light (2.4 × 1014 photons s−1 cm−2) was turned on 1.3 s before the change in Vm. (B) Effect of varying the size of the voltage step. A different photoreceptor was stimulated with a similar protocol, except that the voltage was stepped to −50, −60, −70, and −80 mV on succeeding trials. In divalent-free solution, the current change after the voltage step had a nearly rectangular time course and an amplitude proportional to the voltage stimulus. In the presence of Ca2+, the current swiftly decayed to a small-amplitude plateau level that was graded with Vm. A qualitatively similar effect occurred with Mg2+, but the effect was much reduced, both in terms of relaxation amplitude and speed. The dotted lines mark the 0-current level. (C) Plot of the time constant obtained by fitting single exponential functions to the relaxations measured in 60 mM Ca2+, in response to steps to different voltages from a holding potential of 0 mV. Each point is the average of three cells; error bars represent standard deviations.
Mentions: Considering the disparate potency of voltage-dependent block by Ca2+ and Mg2+ at steady holding potentials (Fig. 2), some mechanistic insight can be gained by comparing their respective kinetics by perturbation/relaxation analysis. Fig. 8 A shows the normalized currents evoked by a hyperpolarizing step from 0 to −70 mV during illumination, in a cell that was successively superfused with extracellular solution containing elevated K (50 mM), either devoid of divalents or in the presence of 60 mM Ca2+ or 60 mM Mg2+. As before, in 0-divalents, the photocurrent remained stationary after the voltage perturbation, whereas a conspicuous relaxation occurred in the presence of either divalent cation; the most striking difference, however, is that the time course with calcium (τ = 7 ms) was much faster than with magnesium (τ = 29 ms). In both ionic conditions, the relaxations accelerated as a function of the membrane hyperpolarization: in Fig. 8 B, the voltage was stepped in 10-mV increments between −50 and −80 mV, from a holding potential of 0 mV. It is clear that both the amplitude and the speed of the relaxation are graded with the size of the voltage stimulus, although with Mg2+ these transients remained significantly smaller and slower. By contrast, in 0-divalents, the currents after each step retained a nearly flat time course (except for some slow creep), and their amplitude changed linearly with voltage. These effects were confirmed in a total of five cells (two tested with a shortened protocol). The range of voltages examined could not be extended further, because the applied stimulus had to be significantly more negative than approximately −40 mV to insure a reasonable driving force, but not too large, otherwise membrane breakdown occurs (Gomez and Nasi 1994a), a situation that is exacerbated by removal of divalents (notice the noisy traces in Fig. 1 A and 7 B). The progressive shortening of the relaxation time constant with membrane hyperpolarization in the presence of Ca2+ is illustrated in Fig. 8 C; each point in the plot represents an average value (n = 3). The corresponding data for magnesium (not shown) were more complex in that the sum of two exponential functions was often required to satisfactorily fit the data, with the relative contribution of the two components changing significantly as a function of voltage.

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