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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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Demonstration of trapping of blocking divalents within light-sensitive channels. (A) Light steps lasting 1 s were delivered 30-s apart to a photoreceptor cell voltage clamped at 0 mV. During presentation of the first stimulus, the voltage was abruptly stepped to −70 mV until ≈5 s after light termination, after which it was returned to the holding level of 0 mV. The response to the next light exhibits a noticeable delay in its activation. This effect could be replicated by alternating trials with and without the conditioning hyperpolarizing voltage step. (B) Expanded view of the rising phase of the photocurrent, to highlight the temporal lag between the two normalized traces. (C) Superimposed control photocurrents obtained with four repetitions of the light stimulus, without conditioning voltage steps. Light intensity 3.5 × 1014 photons s−1 cm−2.
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Figure 14: Demonstration of trapping of blocking divalents within light-sensitive channels. (A) Light steps lasting 1 s were delivered 30-s apart to a photoreceptor cell voltage clamped at 0 mV. During presentation of the first stimulus, the voltage was abruptly stepped to −70 mV until ≈5 s after light termination, after which it was returned to the holding level of 0 mV. The response to the next light exhibits a noticeable delay in its activation. This effect could be replicated by alternating trials with and without the conditioning hyperpolarizing voltage step. (B) Expanded view of the rising phase of the photocurrent, to highlight the temporal lag between the two normalized traces. (C) Superimposed control photocurrents obtained with four repetitions of the light stimulus, without conditioning voltage steps. Light intensity 3.5 × 1014 photons s−1 cm−2.

Mentions: A final question concerns the fate of a blocking ion upon cessation of photostimulation. Either the gate has to wait for the divalent to vacate the site before closing (owing to some steric hindrance) or, alternatively, the channel could close with the divalent bound within the pore. In the latter case, the fact that calcium and magnesium are not measurably permeant precludes the possibility of any significant fluxing to the cytosol, and so the ion would remain trapped. Appropriate tests to reveal either phenomenon are conceptually straightforward, but achieving the necessary sensitivity with a low-affinity blocker can be arduous. For the “foot in the door” case, one would expect the blocker to slow down the falling phase of the photocurrent; however, because this time constant is already on the order of hundreds of milliseconds, the unblock kinetics would be unlikely to make any significant contribution. In case trapping occurs, if one induced the channels to close during strong blockade, the response to a subsequent light delivered under conditions of reduced block would be expected to have a delayed onset, as the blocker would have to leave its site before current can flow. Because in Pecten ciliary photoreceptors the rising phase of the photocurrent elicited by a bright stimulus is swift and highly reproducible, the possibility exists, in principle, that this effect may be detectable. The results of such an experiment are shown in Fig. 14. A photoreceptor was voltage clamped at 0 mV and stimulated every 30 s with a light step lasting 1 s. On alternating trials, the voltage was abruptly stepped to −70 mV during presentation of the light in order to greatly enhance blockade by divalents; the negative Vm was maintained for ≈5 s after light termination, before being gradually returned to the holding level of 0 mV. The subsequent light may thus activate channels while still in a blocked state, and, upon opening, blockade would take milliseconds to reequilibrate at 0 mV. Alternating stimuli either not preceded by the trapping hyperpolarization or in which the hyperpolarizing step ended before the light termination provided a suitable control. Two superimposed traces obtained with this protocol are shown in Fig. 14 A: the photocurrent that had been preceded by a trapping voltage stimulus during the previous light stimulus exhibited a slight temporal lag with respect to the control record. This difference is more clearly visible in B, where the rising phase of the response is shown in an expanded time scale; the phenomenon could be reproduced with successive repetitions of the protocol. In the same cell, control trials in which the light was presented alone gave rise to photocurrents with virtually identical kinetics (C). The effect was observed in six of nine cells tested, and the average temporal lag, measured at half-maximal response amplitude, was 3.6 ± 2.2 ms.


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)

Demonstration of trapping of blocking divalents within light-sensitive channels. (A) Light steps lasting 1 s were delivered 30-s apart to a photoreceptor cell voltage clamped at 0 mV. During presentation of the first stimulus, the voltage was abruptly stepped to −70 mV until ≈5 s after light termination, after which it was returned to the holding level of 0 mV. The response to the next light exhibits a noticeable delay in its activation. This effect could be replicated by alternating trials with and without the conditioning hyperpolarizing voltage step. (B) Expanded view of the rising phase of the photocurrent, to highlight the temporal lag between the two normalized traces. (C) Superimposed control photocurrents obtained with four repetitions of the light stimulus, without conditioning voltage steps. Light intensity 3.5 × 1014 photons s−1 cm−2.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2230541&req=5

Figure 14: Demonstration of trapping of blocking divalents within light-sensitive channels. (A) Light steps lasting 1 s were delivered 30-s apart to a photoreceptor cell voltage clamped at 0 mV. During presentation of the first stimulus, the voltage was abruptly stepped to −70 mV until ≈5 s after light termination, after which it was returned to the holding level of 0 mV. The response to the next light exhibits a noticeable delay in its activation. This effect could be replicated by alternating trials with and without the conditioning hyperpolarizing voltage step. (B) Expanded view of the rising phase of the photocurrent, to highlight the temporal lag between the two normalized traces. (C) Superimposed control photocurrents obtained with four repetitions of the light stimulus, without conditioning voltage steps. Light intensity 3.5 × 1014 photons s−1 cm−2.
Mentions: A final question concerns the fate of a blocking ion upon cessation of photostimulation. Either the gate has to wait for the divalent to vacate the site before closing (owing to some steric hindrance) or, alternatively, the channel could close with the divalent bound within the pore. In the latter case, the fact that calcium and magnesium are not measurably permeant precludes the possibility of any significant fluxing to the cytosol, and so the ion would remain trapped. Appropriate tests to reveal either phenomenon are conceptually straightforward, but achieving the necessary sensitivity with a low-affinity blocker can be arduous. For the “foot in the door” case, one would expect the blocker to slow down the falling phase of the photocurrent; however, because this time constant is already on the order of hundreds of milliseconds, the unblock kinetics would be unlikely to make any significant contribution. In case trapping occurs, if one induced the channels to close during strong blockade, the response to a subsequent light delivered under conditions of reduced block would be expected to have a delayed onset, as the blocker would have to leave its site before current can flow. Because in Pecten ciliary photoreceptors the rising phase of the photocurrent elicited by a bright stimulus is swift and highly reproducible, the possibility exists, in principle, that this effect may be detectable. The results of such an experiment are shown in Fig. 14. A photoreceptor was voltage clamped at 0 mV and stimulated every 30 s with a light step lasting 1 s. On alternating trials, the voltage was abruptly stepped to −70 mV during presentation of the light in order to greatly enhance blockade by divalents; the negative Vm was maintained for ≈5 s after light termination, before being gradually returned to the holding level of 0 mV. The subsequent light may thus activate channels while still in a blocked state, and, upon opening, blockade would take milliseconds to reequilibrate at 0 mV. Alternating stimuli either not preceded by the trapping hyperpolarization or in which the hyperpolarizing step ended before the light termination provided a suitable control. Two superimposed traces obtained with this protocol are shown in Fig. 14 A: the photocurrent that had been preceded by a trapping voltage stimulus during the previous light stimulus exhibited a slight temporal lag with respect to the control record. This difference is more clearly visible in B, where the rising phase of the response is shown in an expanded time scale; the phenomenon could be reproduced with successive repetitions of the protocol. In the same cell, control trials in which the light was presented alone gave rise to photocurrents with virtually identical kinetics (C). The effect was observed in six of nine cells tested, and the average temporal lag, measured at half-maximal response amplitude, was 3.6 ± 2.2 ms.

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