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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 blockade of the photocurrent by Ca2+ and by Mg2+. A photoreceptor was voltage clamped at −30 mV and stimulated repetitively with 100-ms flashes (2.4 × 1014 photons s−1 cm−2) delivered every minute while superfusing with 0-divalents ASW, or in the presence of 60 mM Mg2+ or 60 mM Ca2+. Both divalents reduced the light response amplitude, but the effect was substantially greater for calcium. (B) Rectification of the photocurrent induced by Ca2+ or by Mg2+. The I–V current for the light-evoked current was measured in 10-mV increments in 0-divalents ASW (▪), 60 mM Mg2+ (□), or 60 mM Ca 2+ (▵). Each point represents the average of five cells: peak current amplitudes for each cell were normalized with respect to the value measured at +20 mV; error bars represent standard deviation. The same light intensity as in A was used throughout.
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Figure 2: Comparison of the blockade of the photocurrent by Ca2+ and by Mg2+. A photoreceptor was voltage clamped at −30 mV and stimulated repetitively with 100-ms flashes (2.4 × 1014 photons s−1 cm−2) delivered every minute while superfusing with 0-divalents ASW, or in the presence of 60 mM Mg2+ or 60 mM Ca2+. Both divalents reduced the light response amplitude, but the effect was substantially greater for calcium. (B) Rectification of the photocurrent induced by Ca2+ or by Mg2+. The I–V current for the light-evoked current was measured in 10-mV increments in 0-divalents ASW (▪), 60 mM Mg2+ (□), or 60 mM Ca 2+ (▵). Each point represents the average of five cells: peak current amplitudes for each cell were normalized with respect to the value measured at +20 mV; error bars represent standard deviation. The same light intensity as in A was used throughout.

Mentions: To determine whether both Ca2+ and Mg2+ are capable of blocking the channel, we tested each divalent cation individually. Fig. 2 A shows photocurrents elicited at −30 mV by repetitive flashes delivered initially in 0-divalents extracellular solution, and subsequently after introducing either 60 mM Ca2+ or Mg2+. The amplitude of the light response was reduced in both cases, but the effect was significantly greater for calcium (82% ± 3%, n = 5) than for magnesium (31% ± 6%, n = 3). The average normalized I–V curves measured between −60 and +20 mV in the three conditions is shown in Fig. 2 B (n = 5 per group). Each test was preceded and followed by a control flash at −30 mV; cells that failed to satisfy the criterion of <5% change between the pre- and post-test responses were discarded. It is readily apparent that, although Mg2+ induced a significant curvature in the I–V relation (especially evident at Vm < −40 mV), the outward rectification induced by Ca2+ was far more pronounced. The greater relative potency of Ca2+ vs. Mg2+ was also corroborated by the observation that, when the solution is changed from normal to 0-Ca ASW (by substituting Ca2+ with Mg2+ on an equimolar basis), the photocurrent amplitude is increased and the outward rectification is substantially attenuated (n = 3; data not shown); conversely, a switch from normal to 0-Mg ASW (60 Ca2+) leads to a reduction in the photoresponse (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)

Comparison of the blockade of the photocurrent by Ca2+ and by Mg2+. A photoreceptor was voltage clamped at −30 mV and stimulated repetitively with 100-ms flashes (2.4 × 1014 photons s−1 cm−2) delivered every minute while superfusing with 0-divalents ASW, or in the presence of 60 mM Mg2+ or 60 mM Ca2+. Both divalents reduced the light response amplitude, but the effect was substantially greater for calcium. (B) Rectification of the photocurrent induced by Ca2+ or by Mg2+. The I–V current for the light-evoked current was measured in 10-mV increments in 0-divalents ASW (▪), 60 mM Mg2+ (□), or 60 mM Ca 2+ (▵). Each point represents the average of five cells: peak current amplitudes for each cell were normalized with respect to the value measured at +20 mV; error bars represent standard deviation. The same light intensity as in A was used throughout.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2230541&req=5

Figure 2: Comparison of the blockade of the photocurrent by Ca2+ and by Mg2+. A photoreceptor was voltage clamped at −30 mV and stimulated repetitively with 100-ms flashes (2.4 × 1014 photons s−1 cm−2) delivered every minute while superfusing with 0-divalents ASW, or in the presence of 60 mM Mg2+ or 60 mM Ca2+. Both divalents reduced the light response amplitude, but the effect was substantially greater for calcium. (B) Rectification of the photocurrent induced by Ca2+ or by Mg2+. The I–V current for the light-evoked current was measured in 10-mV increments in 0-divalents ASW (▪), 60 mM Mg2+ (□), or 60 mM Ca 2+ (▵). Each point represents the average of five cells: peak current amplitudes for each cell were normalized with respect to the value measured at +20 mV; error bars represent standard deviation. The same light intensity as in A was used throughout.
Mentions: To determine whether both Ca2+ and Mg2+ are capable of blocking the channel, we tested each divalent cation individually. Fig. 2 A shows photocurrents elicited at −30 mV by repetitive flashes delivered initially in 0-divalents extracellular solution, and subsequently after introducing either 60 mM Ca2+ or Mg2+. The amplitude of the light response was reduced in both cases, but the effect was significantly greater for calcium (82% ± 3%, n = 5) than for magnesium (31% ± 6%, n = 3). The average normalized I–V curves measured between −60 and +20 mV in the three conditions is shown in Fig. 2 B (n = 5 per group). Each test was preceded and followed by a control flash at −30 mV; cells that failed to satisfy the criterion of <5% change between the pre- and post-test responses were discarded. It is readily apparent that, although Mg2+ induced a significant curvature in the I–V relation (especially evident at Vm < −40 mV), the outward rectification induced by Ca2+ was far more pronounced. The greater relative potency of Ca2+ vs. Mg2+ was also corroborated by the observation that, when the solution is changed from normal to 0-Ca ASW (by substituting Ca2+ with Mg2+ on an equimolar basis), the photocurrent amplitude is increased and the outward rectification is substantially attenuated (n = 3; data not shown); conversely, a switch from normal to 0-Mg ASW (60 Ca2+) leads to a reduction in the photoresponse (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