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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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Effect of divalent concentration on the voltage-induced photocurrent transients. (A) Relaxations measured with hyperpolarizing voltage steps from 0 to −80 mV in a photoreceptor cell bathed in high-K solution containing 2, 10, or 60 mM calcium (no Mg2+). The speed and amplitude of the relaxation increased in a manner graded with [Ca2+]o. (B) Normalized traces from A, underscoring the progressive acceleration of the enhancement of blockade induced by perturbations of Vm, as extracellular Ca2+ was raised. Light intensity 3.5 × 1014 photons s−1 cm−2. (C) Apparent association (▪) and dissociation (□) rates for calcium, calculated from the average relaxation time constants and steady state blockade in the presence of the three Ca2+ concentrations (n = 3). The circles represent the corresponding parameters for magnesium, estimated at 60 mM.
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Figure 9: Effect of divalent concentration on the voltage-induced photocurrent transients. (A) Relaxations measured with hyperpolarizing voltage steps from 0 to −80 mV in a photoreceptor cell bathed in high-K solution containing 2, 10, or 60 mM calcium (no Mg2+). The speed and amplitude of the relaxation increased in a manner graded with [Ca2+]o. (B) Normalized traces from A, underscoring the progressive acceleration of the enhancement of blockade induced by perturbations of Vm, as extracellular Ca2+ was raised. Light intensity 3.5 × 1014 photons s−1 cm−2. (C) Apparent association (▪) and dissociation (□) rates for calcium, calculated from the average relaxation time constants and steady state blockade in the presence of the three Ca2+ concentrations (n = 3). The circles represent the corresponding parameters for magnesium, estimated at 60 mM.

Mentions: The dependency of the relaxation parameters on the concentration of the blocking ion is illustrated in Fig. 9 A. A photoreceptor was voltage clamped at 0 mV and stimulated with a step to −80 mV; the test was repeated using different concentrations of Ca2+ (2, 10, and 60 mM) as the sole extracellular divalent cation. Increasing calcium had two clear effects: (a) it resulted in a smaller steady state current, and (b) it accelerated the time course of the relaxation. The latter result is highlighted by Fig. 9 B, in which the relaxations measured at different [Ca2+]o were normalized and superimposed. Similar observations were made in three cells. Notice that the initial peak amplitude of the transient was also inversely related to the Ca2+ concentration, owing to the fact that, at the holding voltage of 0 mV, a significant degree of blockade is already present (e.g., ≈50% for 60 mM Ca2+; see Fig. 3 C). In Fig. 9 C, the kinetic rates were estimated after pooling the data obtained across different photoreceptors; assuming one-to-one interaction, these can be derived from the standard relations: τ = 1/(α × [D] + β) and Kd = β/α, where τ is the measured time constant of the relaxation, [D] is the divalent concentration, and the apparent Kd was determined from the reduction of photocurrent amplitude upon introducing the divalents at a steady Vm (−80 mV). α are β are the forward and reverse rate constants, respectively. As expected, the “off” rate (□) was independent of [Ca2+]o (≈11 s−1), whereas the apparent association rate (▪) increased as a function of the concentration of the blocking ion. The plot also includes the corresponding values obtained with 60 mM Mg2+ (○; average of n = 3). It is noteworthy that the intrinsic “on” rate constant for Mg2+ (≈6.8 × 102 M−1 s−1) was substantially lower than that for Ca2+ (≈3.7 × 103 M−1 s−1 from the fitted line).


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)

Effect of divalent concentration on the voltage-induced photocurrent transients. (A) Relaxations measured with hyperpolarizing voltage steps from 0 to −80 mV in a photoreceptor cell bathed in high-K solution containing 2, 10, or 60 mM calcium (no Mg2+). The speed and amplitude of the relaxation increased in a manner graded with [Ca2+]o. (B) Normalized traces from A, underscoring the progressive acceleration of the enhancement of blockade induced by perturbations of Vm, as extracellular Ca2+ was raised. Light intensity 3.5 × 1014 photons s−1 cm−2. (C) Apparent association (▪) and dissociation (□) rates for calcium, calculated from the average relaxation time constants and steady state blockade in the presence of the three Ca2+ concentrations (n = 3). The circles represent the corresponding parameters for magnesium, estimated at 60 mM.
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Related In: Results  -  Collection

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Figure 9: Effect of divalent concentration on the voltage-induced photocurrent transients. (A) Relaxations measured with hyperpolarizing voltage steps from 0 to −80 mV in a photoreceptor cell bathed in high-K solution containing 2, 10, or 60 mM calcium (no Mg2+). The speed and amplitude of the relaxation increased in a manner graded with [Ca2+]o. (B) Normalized traces from A, underscoring the progressive acceleration of the enhancement of blockade induced by perturbations of Vm, as extracellular Ca2+ was raised. Light intensity 3.5 × 1014 photons s−1 cm−2. (C) Apparent association (▪) and dissociation (□) rates for calcium, calculated from the average relaxation time constants and steady state blockade in the presence of the three Ca2+ concentrations (n = 3). The circles represent the corresponding parameters for magnesium, estimated at 60 mM.
Mentions: The dependency of the relaxation parameters on the concentration of the blocking ion is illustrated in Fig. 9 A. A photoreceptor was voltage clamped at 0 mV and stimulated with a step to −80 mV; the test was repeated using different concentrations of Ca2+ (2, 10, and 60 mM) as the sole extracellular divalent cation. Increasing calcium had two clear effects: (a) it resulted in a smaller steady state current, and (b) it accelerated the time course of the relaxation. The latter result is highlighted by Fig. 9 B, in which the relaxations measured at different [Ca2+]o were normalized and superimposed. Similar observations were made in three cells. Notice that the initial peak amplitude of the transient was also inversely related to the Ca2+ concentration, owing to the fact that, at the holding voltage of 0 mV, a significant degree of blockade is already present (e.g., ≈50% for 60 mM Ca2+; see Fig. 3 C). In Fig. 9 C, the kinetic rates were estimated after pooling the data obtained across different photoreceptors; assuming one-to-one interaction, these can be derived from the standard relations: τ = 1/(α × [D] + β) and Kd = β/α, where τ is the measured time constant of the relaxation, [D] is the divalent concentration, and the apparent Kd was determined from the reduction of photocurrent amplitude upon introducing the divalents at a steady Vm (−80 mV). α are β are the forward and reverse rate constants, respectively. As expected, the “off” rate (□) was independent of [Ca2+]o (≈11 s−1), whereas the apparent association rate (▪) increased as a function of the concentration of the blocking ion. The plot also includes the corresponding values obtained with 60 mM Mg2+ (○; average of n = 3). It is noteworthy that the intrinsic “on” rate constant for Mg2+ (≈6.8 × 102 M−1 s−1) was substantially lower than that for Ca2+ (≈3.7 × 103 M−1 s−1 from the fitted line).

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