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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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Light-dependent single-channel currents are resolvable in the presence of divalent cations. (A) Example of cell-attached recording in the ciliary appendages of a hyperpolarizing photoreceptor cell, under normal ionic conditions. The pipette contained ASW and was depolarized by −90 mV. A step of light (3 × 1016 photons s−1 cm−2) repeatedly applied for 2 s at 1-min intervals elicited outward channel openings; no activity was observed in the dark. (B) Light-evoked unitary currents can also be resolved at negative potentials, provided that conditions are arranged to ensure a suitable driving force. The recording was obtained at −30 mV electrode potential, with a filling solution containing 490 mM KCl (in addition to the normal concentration of divalent cations). A step of light (4.4 × 1014 photons s−1 cm−2) evoked distinct channel openings in the inward direction (top). (Inset) A 40-ms stretch of recording, marked by the star, is shown on an expanded time scale to illustrate the lack of rapid flickering. A 50-mV hyperpolarizing voltage-step applied in the dark to mimic the receptor potential produced no channel events (bottom).
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Figure 5: Light-dependent single-channel currents are resolvable in the presence of divalent cations. (A) Example of cell-attached recording in the ciliary appendages of a hyperpolarizing photoreceptor cell, under normal ionic conditions. The pipette contained ASW and was depolarized by −90 mV. A step of light (3 × 1016 photons s−1 cm−2) repeatedly applied for 2 s at 1-min intervals elicited outward channel openings; no activity was observed in the dark. (B) Light-evoked unitary currents can also be resolved at negative potentials, provided that conditions are arranged to ensure a suitable driving force. The recording was obtained at −30 mV electrode potential, with a filling solution containing 490 mM KCl (in addition to the normal concentration of divalent cations). A step of light (4.4 × 1014 photons s−1 cm−2) evoked distinct channel openings in the inward direction (top). (Inset) A 40-ms stretch of recording, marked by the star, is shown on an expanded time scale to illustrate the lack of rapid flickering. A 50-mV hyperpolarizing voltage-step applied in the dark to mimic the receptor potential produced no channel events (bottom).

Mentions: Unlike in vertebrate rods, in Pecten ciliary photoreceptors, light-dependent single-channel currents can be resolved in the presence of normal concentrations of extracellular Ca2+ and Mg2+ (Gomez and Nasi 1994a). Fig. 5 A illustrates an example obtained in a cell-attached patch from a ciliary appendage, with the pipette filled with normal ASW and depolarized by 90 mV to produce a suitable driving force. Steps of light lasting for 2 s repeatedly administered at 1-min intervals elicited distinct outward single-channel currents. Because the intensity of the light was saturating, the receptor potential may have approached −80 mV at the peak of the response (Gorman and McReynolds 1978), bringing the absolute voltage across the patch somewhat positive of 0 mV. At that membrane potential, the effect of divalents is inevitably greatly reduced (see Fig. 1 A and 3 D); therefore, additional measurements were performed at more negative voltages where blockade is far more substantial. To that end, a pipette solution containing elevated potassium (490 mM, replacing Na on an equimolar basis) was used; the resulting estimated value of EK is near +12 mV, which ensures a sizable inwardly directed driving force on K ions over the voltage range of interest. Fig. 5 B shows that with Vp set at −30 mV, light-activated single-channel currents could still be recorded. Application of a hyperpolarizing voltage step in the dark to mimic the receptor potential failed to evoke any openings, confirming that channel activation was not a consequence of the change in Vm. Two additional patches tested under the same conditions yielded similar results; when light stimulation was delivered at Vp = 0 mV (i.e., with the physiological potential across the patch), unitary currents were again clearly resolved.


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)

Light-dependent single-channel currents are resolvable in the presence of divalent cations. (A) Example of cell-attached recording in the ciliary appendages of a hyperpolarizing photoreceptor cell, under normal ionic conditions. The pipette contained ASW and was depolarized by −90 mV. A step of light (3 × 1016 photons s−1 cm−2) repeatedly applied for 2 s at 1-min intervals elicited outward channel openings; no activity was observed in the dark. (B) Light-evoked unitary currents can also be resolved at negative potentials, provided that conditions are arranged to ensure a suitable driving force. The recording was obtained at −30 mV electrode potential, with a filling solution containing 490 mM KCl (in addition to the normal concentration of divalent cations). A step of light (4.4 × 1014 photons s−1 cm−2) evoked distinct channel openings in the inward direction (top). (Inset) A 40-ms stretch of recording, marked by the star, is shown on an expanded time scale to illustrate the lack of rapid flickering. A 50-mV hyperpolarizing voltage-step applied in the dark to mimic the receptor potential produced no channel events (bottom).
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Figure 5: Light-dependent single-channel currents are resolvable in the presence of divalent cations. (A) Example of cell-attached recording in the ciliary appendages of a hyperpolarizing photoreceptor cell, under normal ionic conditions. The pipette contained ASW and was depolarized by −90 mV. A step of light (3 × 1016 photons s−1 cm−2) repeatedly applied for 2 s at 1-min intervals elicited outward channel openings; no activity was observed in the dark. (B) Light-evoked unitary currents can also be resolved at negative potentials, provided that conditions are arranged to ensure a suitable driving force. The recording was obtained at −30 mV electrode potential, with a filling solution containing 490 mM KCl (in addition to the normal concentration of divalent cations). A step of light (4.4 × 1014 photons s−1 cm−2) evoked distinct channel openings in the inward direction (top). (Inset) A 40-ms stretch of recording, marked by the star, is shown on an expanded time scale to illustrate the lack of rapid flickering. A 50-mV hyperpolarizing voltage-step applied in the dark to mimic the receptor potential produced no channel events (bottom).
Mentions: Unlike in vertebrate rods, in Pecten ciliary photoreceptors, light-dependent single-channel currents can be resolved in the presence of normal concentrations of extracellular Ca2+ and Mg2+ (Gomez and Nasi 1994a). Fig. 5 A illustrates an example obtained in a cell-attached patch from a ciliary appendage, with the pipette filled with normal ASW and depolarized by 90 mV to produce a suitable driving force. Steps of light lasting for 2 s repeatedly administered at 1-min intervals elicited distinct outward single-channel currents. Because the intensity of the light was saturating, the receptor potential may have approached −80 mV at the peak of the response (Gorman and McReynolds 1978), bringing the absolute voltage across the patch somewhat positive of 0 mV. At that membrane potential, the effect of divalents is inevitably greatly reduced (see Fig. 1 A and 3 D); therefore, additional measurements were performed at more negative voltages where blockade is far more substantial. To that end, a pipette solution containing elevated potassium (490 mM, replacing Na on an equimolar basis) was used; the resulting estimated value of EK is near +12 mV, which ensures a sizable inwardly directed driving force on K ions over the voltage range of interest. Fig. 5 B shows that with Vp set at −30 mV, light-activated single-channel currents could still be recorded. Application of a hyperpolarizing voltage step in the dark to mimic the receptor potential failed to evoke any openings, confirming that channel activation was not a consequence of the change in Vm. Two additional patches tested under the same conditions yielded similar results; when light stimulation was delivered at Vp = 0 mV (i.e., with the physiological potential across the patch), unitary currents were again clearly resolved.

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