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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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(A) Modulation of the kinetics of the depolarization-activated outward current by light. A photoreceptor was voltage clamped at a holding potential of −80 mV and stimulated with depolarizing steps in 10-mV increments. In the dark (left), part of the outward current is contributed by IA, which confers the characteristic decaying kinetics. In the presence of steady light (2.4 × 1014 photons s−1 cm−2), the time course became sustained (right). (B) Steady state inactivation of the transient component of the outward current in the dark. A ciliary photoreceptor was stimulated by a depolarizing voltage step to 0 mV, preceded by a conditioning step between −10 and −100 mV, lasting for 500 ms (only the last 50 ms are shown). A distinct transient outward current was elicited when the conditioning voltage was substantially negative, but inactivated at more positive voltages. (C) Steady state inactivation curve. The peak amplitude of the outward current at 0 mV from B is plotted as a function of the voltage of the preceding conditioning step. The data points were fitted by a Boltzmann function; the half-maximum inactivation voltage was approximately −67 mV. Recordings were performed in standard ASW.
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Figure 10: (A) Modulation of the kinetics of the depolarization-activated outward current by light. A photoreceptor was voltage clamped at a holding potential of −80 mV and stimulated with depolarizing steps in 10-mV increments. In the dark (left), part of the outward current is contributed by IA, which confers the characteristic decaying kinetics. In the presence of steady light (2.4 × 1014 photons s−1 cm−2), the time course became sustained (right). (B) Steady state inactivation of the transient component of the outward current in the dark. A ciliary photoreceptor was stimulated by a depolarizing voltage step to 0 mV, preceded by a conditioning step between −10 and −100 mV, lasting for 500 ms (only the last 50 ms are shown). A distinct transient outward current was elicited when the conditioning voltage was substantially negative, but inactivated at more positive voltages. (C) Steady state inactivation curve. The peak amplitude of the outward current at 0 mV from B is plotted as a function of the voltage of the preceding conditioning step. The data points were fitted by a Boltzmann function; the half-maximum inactivation voltage was approximately −67 mV. Recordings were performed in standard ASW.

Mentions: To demonstrate the relief of block, symmetrical experiments were conducted in which depolarizing steps were applied from a negative holding voltage, with and without illumination. This procedure, however, requires some caution for the following reason: whereas membrane hyperpolarization elicits no active currents, depolarization can trigger several voltage-dependent mechanisms, including Ca2+ and K channels (Cornwall and Gorman 1979). In principle, none of these would be expected to pose a problem in that the current subtraction protocol should cancel out any contribution by non–light-dependent processes. However, a recent report in distal photoreceptors of a related species of scallop (Patinopecten yessoensis) demonstrated the presence of a transient K current (IA) whose decay kinetics are modulated by light (Shimatani and Katagiri 1995). Fig. 10 A shows that a similar phenomenon also occurs in Pecten irradians: an isolated photoreceptor was voltage clamped at −80 mV and stimulated with depolarizing pulses of increasing amplitude in 10-mV increments. In the dark, voltage steps more positive than −30 mV elicited an outward current consisting of a transient and a sustained component (left); the inactivating current is carried by potassium and blocked by 4-aminopyridine (4-AP, data not shown). When the protocol was repeated in the presence of steady light (right), the time course of the outward current changed markedly, becoming sustained (n = 8). This phenomenon can undermine the validity of subtracting currents elicited by depolarizing pulses in the dark and light because spurious relaxations could be artifactually introduced. Blockade of IA by 4-AP is not a viable option, as we have previously shown that the light-dependent K conductance in these photoreceptor cells is also extraordinarily susceptible to blockade by this drug (Gomez and Nasi 1994b). An alternative strategy is to exploit the steady-state inactivation properties of IA to minimize its contribution: Fig. 9 B shows recordings obtained in the dark in a distal photoreceptor subjected to a 500-ms conditioning prestep to various voltages between −10 and −100 mV, followed by a depolarization to 0 mV. As the conditioning voltage was made more negative, a distinct transient outward current was evoked by the step to 0 mV. In Fig. 9 C, the steady state inactivation data were fitted by a Boltzmann function; the average voltage for half-maximal inactivation was −67 ± 8 mV (n = 4).


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)

(A) Modulation of the kinetics of the depolarization-activated outward current by light. A photoreceptor was voltage clamped at a holding potential of −80 mV and stimulated with depolarizing steps in 10-mV increments. In the dark (left), part of the outward current is contributed by IA, which confers the characteristic decaying kinetics. In the presence of steady light (2.4 × 1014 photons s−1 cm−2), the time course became sustained (right). (B) Steady state inactivation of the transient component of the outward current in the dark. A ciliary photoreceptor was stimulated by a depolarizing voltage step to 0 mV, preceded by a conditioning step between −10 and −100 mV, lasting for 500 ms (only the last 50 ms are shown). A distinct transient outward current was elicited when the conditioning voltage was substantially negative, but inactivated at more positive voltages. (C) Steady state inactivation curve. The peak amplitude of the outward current at 0 mV from B is plotted as a function of the voltage of the preceding conditioning step. The data points were fitted by a Boltzmann function; the half-maximum inactivation voltage was approximately −67 mV. Recordings were performed in standard ASW.
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Related In: Results  -  Collection

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Figure 10: (A) Modulation of the kinetics of the depolarization-activated outward current by light. A photoreceptor was voltage clamped at a holding potential of −80 mV and stimulated with depolarizing steps in 10-mV increments. In the dark (left), part of the outward current is contributed by IA, which confers the characteristic decaying kinetics. In the presence of steady light (2.4 × 1014 photons s−1 cm−2), the time course became sustained (right). (B) Steady state inactivation of the transient component of the outward current in the dark. A ciliary photoreceptor was stimulated by a depolarizing voltage step to 0 mV, preceded by a conditioning step between −10 and −100 mV, lasting for 500 ms (only the last 50 ms are shown). A distinct transient outward current was elicited when the conditioning voltage was substantially negative, but inactivated at more positive voltages. (C) Steady state inactivation curve. The peak amplitude of the outward current at 0 mV from B is plotted as a function of the voltage of the preceding conditioning step. The data points were fitted by a Boltzmann function; the half-maximum inactivation voltage was approximately −67 mV. Recordings were performed in standard ASW.
Mentions: To demonstrate the relief of block, symmetrical experiments were conducted in which depolarizing steps were applied from a negative holding voltage, with and without illumination. This procedure, however, requires some caution for the following reason: whereas membrane hyperpolarization elicits no active currents, depolarization can trigger several voltage-dependent mechanisms, including Ca2+ and K channels (Cornwall and Gorman 1979). In principle, none of these would be expected to pose a problem in that the current subtraction protocol should cancel out any contribution by non–light-dependent processes. However, a recent report in distal photoreceptors of a related species of scallop (Patinopecten yessoensis) demonstrated the presence of a transient K current (IA) whose decay kinetics are modulated by light (Shimatani and Katagiri 1995). Fig. 10 A shows that a similar phenomenon also occurs in Pecten irradians: an isolated photoreceptor was voltage clamped at −80 mV and stimulated with depolarizing pulses of increasing amplitude in 10-mV increments. In the dark, voltage steps more positive than −30 mV elicited an outward current consisting of a transient and a sustained component (left); the inactivating current is carried by potassium and blocked by 4-aminopyridine (4-AP, data not shown). When the protocol was repeated in the presence of steady light (right), the time course of the outward current changed markedly, becoming sustained (n = 8). This phenomenon can undermine the validity of subtracting currents elicited by depolarizing pulses in the dark and light because spurious relaxations could be artifactually introduced. Blockade of IA by 4-AP is not a viable option, as we have previously shown that the light-dependent K conductance in these photoreceptor cells is also extraordinarily susceptible to blockade by this drug (Gomez and Nasi 1994b). An alternative strategy is to exploit the steady-state inactivation properties of IA to minimize its contribution: Fig. 9 B shows recordings obtained in the dark in a distal photoreceptor subjected to a 500-ms conditioning prestep to various voltages between −10 and −100 mV, followed by a depolarization to 0 mV. As the conditioning voltage was made more negative, a distinct transient outward current was evoked by the step to 0 mV. In Fig. 9 C, the steady state inactivation data were fitted by a Boltzmann function; the average voltage for half-maximal inactivation was −67 ± 8 mV (n = 4).

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