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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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Divalents act like negligibly permeant blockers of the light-dependent conductance. (A) A ciliary photoreceptor was voltage clamped and stimulated with 100-ms flashes of light (9.5 × 1014 photons s−1 cm−2) spaced 1-min apart. On successive trials, the holding potential was stepped in 10-mV increments ≈5 s before the flash. The procedure was carried out in ASW (left), then in divalent-free solution (middle), and back to control (right). At the more negative voltages, light-evoked currents were minute in ASW, but became conspicuous after removal of extracellular Ca2+ and Mg2+. (B) Peak amplitude of the photocurrent plotted as a function of membrane voltage in ASW (▪), 0-divalent solution (▴), and wash (□). Omission of Ca2+ and Mg2+ removed the outward rectification, making the I–V relation essentially linear. (C) Reversal of the photocurrent in high-K solution, in the presence and absence of divalents. Membrane voltage was varied in 2-mV increments between −32 and −48 mV; light intensity was 2.4 × 1014 photons s−1 cm−2. (D) Lack of shift in Vrev upon removing extracellular Ca2+ and Mg2+. Data from three photoreceptor cells tested as in C were pooled; error bars indicate standard deviation.
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Figure 1: Divalents act like negligibly permeant blockers of the light-dependent conductance. (A) A ciliary photoreceptor was voltage clamped and stimulated with 100-ms flashes of light (9.5 × 1014 photons s−1 cm−2) spaced 1-min apart. On successive trials, the holding potential was stepped in 10-mV increments ≈5 s before the flash. The procedure was carried out in ASW (left), then in divalent-free solution (middle), and back to control (right). At the more negative voltages, light-evoked currents were minute in ASW, but became conspicuous after removal of extracellular Ca2+ and Mg2+. (B) Peak amplitude of the photocurrent plotted as a function of membrane voltage in ASW (▪), 0-divalent solution (▴), and wash (□). Omission of Ca2+ and Mg2+ removed the outward rectification, making the I–V relation essentially linear. (C) Reversal of the photocurrent in high-K solution, in the presence and absence of divalents. Membrane voltage was varied in 2-mV increments between −32 and −48 mV; light intensity was 2.4 × 1014 photons s−1 cm−2. (D) Lack of shift in Vrev upon removing extracellular Ca2+ and Mg2+. Data from three photoreceptor cells tested as in C were pooled; error bars indicate standard deviation.

Mentions: In vertebrate photoreceptors, the light-sensitive conductance is poorly selective among cations, and exhibits a substantial permeability to calcium ions (Capovilla et al. 1983; Hodgkin et al. 1984), which carry a significant fraction of the dark current under physiological conditions (Yau and Nakatani 1985; Nakatani and Yau 1988; Perry and McNaughton 1991; Haynes 1995). “Blockade” by Ca2+ reflects the prolonged dwell times in the permeation pathway (presumed to be single file) due to tight binding. In ciliary invertebrate photoreceptors, by contrast, the contribution of divalents to the photocurrent is expected to be negligible because the reversal potential is near EK and changes with a near-perfectly Nernstian dependency upon manipulating [K]o, even in the presence of normal concentrations of extracellular Ca2+ and Mg2+ (Gomez and Nasi 1994a). The experiments illustrated in Fig. 1 were designed to assess in a direct way the permeation of Ca2+ and Mg2+ through light-activated channels. In Fig. 1 A, the membrane potential of a photoreceptor was stepped in 10-mV increments for several seconds before stimulating with a flash of light of fixed intensity; Vm was returned to −30 mV between trials. In normal ASW, the peak amplitude of the photocurrent decreased in a markedly nonlinear way with hyperpolarization, and at −100 mV a barely detectable inward photocurrent was elicited (left). After switching to a solution lacking both calcium and magnesium (middle), the amplitude of the light responses significantly increased, especially at hyperpolarized voltages. In particular, below the reversal potential, a sizable inward photocurrent could be clearly observed. This effect was fully reversible (right). The I–V relation for the three phases of the experiment, plotted in Fig. 1 B, shows that removal of divalents virtually eliminated the rectification, without any obvious concomitant displacement of the reversal potential, which remained near −80 mV (n = 3). To provide a more sensitive test, additional measurements were conducted in the presence of elevated [K]o (50 mM), as shown in Fig. 1 C. These conditions were designed to displace Vrev to a range in which gL is larger so that the greater slope of the I–V curve improves the signal-to-noise ratio (S/N); in addition, the accuracy of the measurements was increased by changing the holding potential in 2-mV increments to detect any small change in the reversal voltage. As shown in Fig. 1 D, Vrev was not significantly affected by the presence or absence of divalent cations (mean shift 0.8 ± 0.3 mV, n = 3), confirming that the permeability of Ca2+ and Mg2+ must be negligible.


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)

Divalents act like negligibly permeant blockers of the light-dependent conductance. (A) A ciliary photoreceptor was voltage clamped and stimulated with 100-ms flashes of light (9.5 × 1014 photons s−1 cm−2) spaced 1-min apart. On successive trials, the holding potential was stepped in 10-mV increments ≈5 s before the flash. The procedure was carried out in ASW (left), then in divalent-free solution (middle), and back to control (right). At the more negative voltages, light-evoked currents were minute in ASW, but became conspicuous after removal of extracellular Ca2+ and Mg2+. (B) Peak amplitude of the photocurrent plotted as a function of membrane voltage in ASW (▪), 0-divalent solution (▴), and wash (□). Omission of Ca2+ and Mg2+ removed the outward rectification, making the I–V relation essentially linear. (C) Reversal of the photocurrent in high-K solution, in the presence and absence of divalents. Membrane voltage was varied in 2-mV increments between −32 and −48 mV; light intensity was 2.4 × 1014 photons s−1 cm−2. (D) Lack of shift in Vrev upon removing extracellular Ca2+ and Mg2+. Data from three photoreceptor cells tested as in C were pooled; error bars indicate standard deviation.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2230541&req=5

Figure 1: Divalents act like negligibly permeant blockers of the light-dependent conductance. (A) A ciliary photoreceptor was voltage clamped and stimulated with 100-ms flashes of light (9.5 × 1014 photons s−1 cm−2) spaced 1-min apart. On successive trials, the holding potential was stepped in 10-mV increments ≈5 s before the flash. The procedure was carried out in ASW (left), then in divalent-free solution (middle), and back to control (right). At the more negative voltages, light-evoked currents were minute in ASW, but became conspicuous after removal of extracellular Ca2+ and Mg2+. (B) Peak amplitude of the photocurrent plotted as a function of membrane voltage in ASW (▪), 0-divalent solution (▴), and wash (□). Omission of Ca2+ and Mg2+ removed the outward rectification, making the I–V relation essentially linear. (C) Reversal of the photocurrent in high-K solution, in the presence and absence of divalents. Membrane voltage was varied in 2-mV increments between −32 and −48 mV; light intensity was 2.4 × 1014 photons s−1 cm−2. (D) Lack of shift in Vrev upon removing extracellular Ca2+ and Mg2+. Data from three photoreceptor cells tested as in C were pooled; error bars indicate standard deviation.
Mentions: In vertebrate photoreceptors, the light-sensitive conductance is poorly selective among cations, and exhibits a substantial permeability to calcium ions (Capovilla et al. 1983; Hodgkin et al. 1984), which carry a significant fraction of the dark current under physiological conditions (Yau and Nakatani 1985; Nakatani and Yau 1988; Perry and McNaughton 1991; Haynes 1995). “Blockade” by Ca2+ reflects the prolonged dwell times in the permeation pathway (presumed to be single file) due to tight binding. In ciliary invertebrate photoreceptors, by contrast, the contribution of divalents to the photocurrent is expected to be negligible because the reversal potential is near EK and changes with a near-perfectly Nernstian dependency upon manipulating [K]o, even in the presence of normal concentrations of extracellular Ca2+ and Mg2+ (Gomez and Nasi 1994a). The experiments illustrated in Fig. 1 were designed to assess in a direct way the permeation of Ca2+ and Mg2+ through light-activated channels. In Fig. 1 A, the membrane potential of a photoreceptor was stepped in 10-mV increments for several seconds before stimulating with a flash of light of fixed intensity; Vm was returned to −30 mV between trials. In normal ASW, the peak amplitude of the photocurrent decreased in a markedly nonlinear way with hyperpolarization, and at −100 mV a barely detectable inward photocurrent was elicited (left). After switching to a solution lacking both calcium and magnesium (middle), the amplitude of the light responses significantly increased, especially at hyperpolarized voltages. In particular, below the reversal potential, a sizable inward photocurrent could be clearly observed. This effect was fully reversible (right). The I–V relation for the three phases of the experiment, plotted in Fig. 1 B, shows that removal of divalents virtually eliminated the rectification, without any obvious concomitant displacement of the reversal potential, which remained near −80 mV (n = 3). To provide a more sensitive test, additional measurements were conducted in the presence of elevated [K]o (50 mM), as shown in Fig. 1 C. These conditions were designed to displace Vrev to a range in which gL is larger so that the greater slope of the I–V curve improves the signal-to-noise ratio (S/N); in addition, the accuracy of the measurements was increased by changing the holding potential in 2-mV increments to detect any small change in the reversal voltage. As shown in Fig. 1 D, Vrev was not significantly affected by the presence or absence of divalent cations (mean shift 0.8 ± 0.3 mV, n = 3), confirming that the permeability of Ca2+ and Mg2+ must be negligible.

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