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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-dependent effects of divalents on the photocurrent kinetics. (A, left) Normalized light responses (100-ms flashes, 9.5 × 1014 photons s−1 cm−2), recorded at different holding voltages between −60 and −20 mV, in 10-mV increments. The time course became more rapid with membrane hyperpolarization. (Right) Photocurrents recorded in the same cell after removal of external Ca2+ and Mg2+: the time course became independent of membrane potential and had a kinetics similar to that obtained at the most depolarized voltages in control conditions. (B) Plot of the photoresponse half-width as a function of membrane potential. The change in the presence of Ca2+ and Mg2+ (▪) is substantially more pronounced than after their removal (□). The two tend to converge at the more depolarized range of values.
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Figure 12: Voltage-dependent effects of divalents on the photocurrent kinetics. (A, left) Normalized light responses (100-ms flashes, 9.5 × 1014 photons s−1 cm−2), recorded at different holding voltages between −60 and −20 mV, in 10-mV increments. The time course became more rapid with membrane hyperpolarization. (Right) Photocurrents recorded in the same cell after removal of external Ca2+ and Mg2+: the time course became independent of membrane potential and had a kinetics similar to that obtained at the most depolarized voltages in control conditions. (B) Plot of the photoresponse half-width as a function of membrane potential. The change in the presence of Ca2+ and Mg2+ (▪) is substantially more pronounced than after their removal (□). The two tend to converge at the more depolarized range of values.

Mentions: One possibility is that the binding sites become available to external divalents only when the light-dependent channels are in the open conformation. A straightforward prediction from this conjecture is that the kinetics of the light response should be affected by the membrane potential imposed at the time of photostimulation. The rationale is that equilibration will only begin as the light-dependent channels gradually open, so that the extent of blockade at different voltages would become fully manifest in the late phase of the photocurrent: the more negative the Vm, the faster the apparent decay of the response. This prediction is borne out by the data shown in Fig. 12. On the left side of Fig. 12 A, photoresponses to a fixed flash were measured in control conditions (ASW), at holding voltages that varied between −60 and −20 mV in 10-mV increments; between trials, the membrane potential was returned to −30 mV. The records were normalized with respect to their peak amplitude. The light response decayed progressively more rapidly as the holding potential was made more negative. To rule out the possibility that the phenomenon may simply be due to a direct effect of voltage on the gating of the light-sensitive channels, the procedure was repeated after superfusing the same cell with divalent-free solution: under these conditions, the flash responses remained virtually superimposable (Fig. 12 A, right), with a slow time course resembling that of the photocurrent in ASW at a depolarized Vm. Shifting the range of voltages tested in ASW by 20 mV in the depolarizing direction, to check for possible effects of surface charge screening, did not alter the differences across the two ionic conditions (not shown). As a simple measure of time course, the response half-width (i.e., the time elapsed between the two crossings of the half-maximal amplitude level) is plotted for the two conditions in Fig. 12 B. In normal ASW, the half-width of the light responses increased progressively with depolarization, approaching the value obtained in 0-divalents, which remained relatively constant. The near invariance of response kinetics in 0-divalents was corroborated in a total of eight cells; the pronounced acceleration of the time course with hyperpolarization in ASW was observed numerous times (n > 20).


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-dependent effects of divalents on the photocurrent kinetics. (A, left) Normalized light responses (100-ms flashes, 9.5 × 1014 photons s−1 cm−2), recorded at different holding voltages between −60 and −20 mV, in 10-mV increments. The time course became more rapid with membrane hyperpolarization. (Right) Photocurrents recorded in the same cell after removal of external Ca2+ and Mg2+: the time course became independent of membrane potential and had a kinetics similar to that obtained at the most depolarized voltages in control conditions. (B) Plot of the photoresponse half-width as a function of membrane potential. The change in the presence of Ca2+ and Mg2+ (▪) is substantially more pronounced than after their removal (□). The two tend to converge at the more depolarized range of values.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2230541&req=5

Figure 12: Voltage-dependent effects of divalents on the photocurrent kinetics. (A, left) Normalized light responses (100-ms flashes, 9.5 × 1014 photons s−1 cm−2), recorded at different holding voltages between −60 and −20 mV, in 10-mV increments. The time course became more rapid with membrane hyperpolarization. (Right) Photocurrents recorded in the same cell after removal of external Ca2+ and Mg2+: the time course became independent of membrane potential and had a kinetics similar to that obtained at the most depolarized voltages in control conditions. (B) Plot of the photoresponse half-width as a function of membrane potential. The change in the presence of Ca2+ and Mg2+ (▪) is substantially more pronounced than after their removal (□). The two tend to converge at the more depolarized range of values.
Mentions: One possibility is that the binding sites become available to external divalents only when the light-dependent channels are in the open conformation. A straightforward prediction from this conjecture is that the kinetics of the light response should be affected by the membrane potential imposed at the time of photostimulation. The rationale is that equilibration will only begin as the light-dependent channels gradually open, so that the extent of blockade at different voltages would become fully manifest in the late phase of the photocurrent: the more negative the Vm, the faster the apparent decay of the response. This prediction is borne out by the data shown in Fig. 12. On the left side of Fig. 12 A, photoresponses to a fixed flash were measured in control conditions (ASW), at holding voltages that varied between −60 and −20 mV in 10-mV increments; between trials, the membrane potential was returned to −30 mV. The records were normalized with respect to their peak amplitude. The light response decayed progressively more rapidly as the holding potential was made more negative. To rule out the possibility that the phenomenon may simply be due to a direct effect of voltage on the gating of the light-sensitive channels, the procedure was repeated after superfusing the same cell with divalent-free solution: under these conditions, the flash responses remained virtually superimposable (Fig. 12 A, right), with a slow time course resembling that of the photocurrent in ASW at a depolarized Vm. Shifting the range of voltages tested in ASW by 20 mV in the depolarizing direction, to check for possible effects of surface charge screening, did not alter the differences across the two ionic conditions (not shown). As a simple measure of time course, the response half-width (i.e., the time elapsed between the two crossings of the half-maximal amplitude level) is plotted for the two conditions in Fig. 12 B. In normal ASW, the half-width of the light responses increased progressively with depolarization, approaching the value obtained in 0-divalents, which remained relatively constant. The near invariance of response kinetics in 0-divalents was corroborated in a total of eight cells; the pronounced acceleration of the time course with hyperpolarization in ASW was observed numerous times (n > 20).

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