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Bimodal control of a Ca(2+)-activated Cl(-) channel by different Ca(2+) signals.

Kuruma A, Hartzell HC - J. Gen. Physiol. (2000)

Bottom Line: At higher [Ca(2+)], the currents did not rectify and were time independent.The deactivation time constants increased linearly with the log of membrane potential.The qualitatively different behaviors of this channel in response to different Ca(2+) concentrations adds a new dimension to Ca(2+) signaling: the same channel can mediate either excitatory or inhibitory responses, depending on the amplitude of the cellular Ca(2+) signal.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, Emory University School of Medicine, Atlanta, Georgia 30322-3030, USA.

ABSTRACT
Ca(2+)-activated Cl(-) channels play important roles in a variety of physiological processes, including epithelial secretion, maintenance of smooth muscle tone, and repolarization of the cardiac action potential. It remains unclear, however, exactly how these channels are controlled by Ca(2+) and voltage. Excised inside-out patches containing many Ca(2+)-activated Cl(-) channels from Xenopus oocytes were used to study channel regulation. The currents were mediated by a single type of Cl(-) channel that exhibited an anionic selectivity of I(-) > Br(-) > Cl(-) (3.6:1.9:1.0), irrespective of the direction of the current flow or [Ca(2+)]. However, depending on the amplitude of the Ca(2+) signal, this channel exhibited qualitatively different behaviors. At [Ca(2+)] < 1 microM, the currents activated slowly upon depolarization and deactivated upon hyperpolarization and the steady state current-voltage relationship was strongly outwardly rectifying. At higher [Ca(2+)], the currents did not rectify and were time independent. This difference in behavior at different [Ca(2+)] was explained by an apparent voltage-dependent Ca(2+) sensitivity of the channel. At +120 mV, the EC(50) for channel activation by Ca(2+) was approximately fourfold less than at -120 mV (0.9 vs. 4 microM). Thus, at [Ca(2+)] < 1 microM, inward current was smaller than outward current and the currents were time dependent as a consequence of voltage-dependent changes in Ca(2+) binding. The voltage-dependent Ca(2+) sensitivity was explained by a kinetic gating scheme in which channel activation was Ca(2+) dependent and channel closing was voltage sensitive. This scheme was supported by the observation that deactivation time constants of currents produced by rapid Ca(2+) concentration jumps were voltage sensitive, but that the activation time constants were Ca(2+) sensitive. The deactivation time constants increased linearly with the log of membrane potential. The qualitatively different behaviors of this channel in response to different Ca(2+) concentrations adds a new dimension to Ca(2+) signaling: the same channel can mediate either excitatory or inhibitory responses, depending on the amplitude of the cellular Ca(2+) signal.

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Kinetic analysis of activation of Ca2+-activated Cl− currents in a representative excised patch. Currents were elicited by voltage-clamp steps applied while the cytosolic face of the patch was exposed to solutions with different free [Ca2+]. The patch was voltage clamped by stepping to various potentials between +200 and +40 mV. The activating phase of the currents were fitted to single exponentials (superimposed) and the time constants were plotted versus membrane potential.
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Figure 9: Kinetic analysis of activation of Ca2+-activated Cl− currents in a representative excised patch. Currents were elicited by voltage-clamp steps applied while the cytosolic face of the patch was exposed to solutions with different free [Ca2+]. The patch was voltage clamped by stepping to various potentials between +200 and +40 mV. The activating phase of the currents were fitted to single exponentials (superimposed) and the time constants were plotted versus membrane potential.

Mentions: We then examined the kinetics of current activation. Depolarizing steps elicited outward currents that exhibited a small instantaneous component followed by a slow activation that took ∼1 s to reach maximum (Fig. 9). Activation of the currents could be fitted with single exponentials, but the fits were often disappointing. Specifically, using least squares algorithms, if the rising phase of the current was well fit, the plateau was usually poorly fit. Using the Pade-LaPlace method (Yeramian and Claverie 1987; Bajzer et al. 1989) for fitting curves to sums of exponentials without a hypothesis about the number of components, one fast exponential component plus another slower nonexponential component were found. Regardless of the method used for fitting the activation, the time constants of the exponential components of the activation usually were weakly influenced by membrane potential or increased with depolarization (Fig. 9 and Fig. 10 A). If channel opening were voltage sensitive, we would expect channel activation to accelerate with depolarization. Although activation was not clearly voltage dependent, activation was Ca2+ dependent: activation became faster with increasing [Ca2+] (Fig. 10 B). We interpret the increased current produced by depolarization at low [Ca2+] as a consequence of a shift in the equilibrium between closed and open states as a result of changing the voltage-sensitive backward rate constant β. The time constant of the approach to the new equilibrium upon depolarization will approximate 1/(α [Ca2+] + β). Thus, as β becomes larger with hyperpolarization, the time constant of activation will become smaller for a constant α [Ca2+]. This is observed with the lowest [Ca2+] in Fig. 10 A. With higher [Ca2+], α [Ca2+] dominates the reaction and the effect of changing β is negligible. As the [Ca2+] becomes greater, the time constant of activation becomes smaller, as expected from the model.


Bimodal control of a Ca(2+)-activated Cl(-) channel by different Ca(2+) signals.

Kuruma A, Hartzell HC - J. Gen. Physiol. (2000)

Kinetic analysis of activation of Ca2+-activated Cl− currents in a representative excised patch. Currents were elicited by voltage-clamp steps applied while the cytosolic face of the patch was exposed to solutions with different free [Ca2+]. The patch was voltage clamped by stepping to various potentials between +200 and +40 mV. The activating phase of the currents were fitted to single exponentials (superimposed) and the time constants were plotted versus membrane potential.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC1887779&req=5

Figure 9: Kinetic analysis of activation of Ca2+-activated Cl− currents in a representative excised patch. Currents were elicited by voltage-clamp steps applied while the cytosolic face of the patch was exposed to solutions with different free [Ca2+]. The patch was voltage clamped by stepping to various potentials between +200 and +40 mV. The activating phase of the currents were fitted to single exponentials (superimposed) and the time constants were plotted versus membrane potential.
Mentions: We then examined the kinetics of current activation. Depolarizing steps elicited outward currents that exhibited a small instantaneous component followed by a slow activation that took ∼1 s to reach maximum (Fig. 9). Activation of the currents could be fitted with single exponentials, but the fits were often disappointing. Specifically, using least squares algorithms, if the rising phase of the current was well fit, the plateau was usually poorly fit. Using the Pade-LaPlace method (Yeramian and Claverie 1987; Bajzer et al. 1989) for fitting curves to sums of exponentials without a hypothesis about the number of components, one fast exponential component plus another slower nonexponential component were found. Regardless of the method used for fitting the activation, the time constants of the exponential components of the activation usually were weakly influenced by membrane potential or increased with depolarization (Fig. 9 and Fig. 10 A). If channel opening were voltage sensitive, we would expect channel activation to accelerate with depolarization. Although activation was not clearly voltage dependent, activation was Ca2+ dependent: activation became faster with increasing [Ca2+] (Fig. 10 B). We interpret the increased current produced by depolarization at low [Ca2+] as a consequence of a shift in the equilibrium between closed and open states as a result of changing the voltage-sensitive backward rate constant β. The time constant of the approach to the new equilibrium upon depolarization will approximate 1/(α [Ca2+] + β). Thus, as β becomes larger with hyperpolarization, the time constant of activation will become smaller for a constant α [Ca2+]. This is observed with the lowest [Ca2+] in Fig. 10 A. With higher [Ca2+], α [Ca2+] dominates the reaction and the effect of changing β is negligible. As the [Ca2+] becomes greater, the time constant of activation becomes smaller, as expected from the model.

Bottom Line: At higher [Ca(2+)], the currents did not rectify and were time independent.The deactivation time constants increased linearly with the log of membrane potential.The qualitatively different behaviors of this channel in response to different Ca(2+) concentrations adds a new dimension to Ca(2+) signaling: the same channel can mediate either excitatory or inhibitory responses, depending on the amplitude of the cellular Ca(2+) signal.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, Emory University School of Medicine, Atlanta, Georgia 30322-3030, USA.

ABSTRACT
Ca(2+)-activated Cl(-) channels play important roles in a variety of physiological processes, including epithelial secretion, maintenance of smooth muscle tone, and repolarization of the cardiac action potential. It remains unclear, however, exactly how these channels are controlled by Ca(2+) and voltage. Excised inside-out patches containing many Ca(2+)-activated Cl(-) channels from Xenopus oocytes were used to study channel regulation. The currents were mediated by a single type of Cl(-) channel that exhibited an anionic selectivity of I(-) > Br(-) > Cl(-) (3.6:1.9:1.0), irrespective of the direction of the current flow or [Ca(2+)]. However, depending on the amplitude of the Ca(2+) signal, this channel exhibited qualitatively different behaviors. At [Ca(2+)] < 1 microM, the currents activated slowly upon depolarization and deactivated upon hyperpolarization and the steady state current-voltage relationship was strongly outwardly rectifying. At higher [Ca(2+)], the currents did not rectify and were time independent. This difference in behavior at different [Ca(2+)] was explained by an apparent voltage-dependent Ca(2+) sensitivity of the channel. At +120 mV, the EC(50) for channel activation by Ca(2+) was approximately fourfold less than at -120 mV (0.9 vs. 4 microM). Thus, at [Ca(2+)] < 1 microM, inward current was smaller than outward current and the currents were time dependent as a consequence of voltage-dependent changes in Ca(2+) binding. The voltage-dependent Ca(2+) sensitivity was explained by a kinetic gating scheme in which channel activation was Ca(2+) dependent and channel closing was voltage sensitive. This scheme was supported by the observation that deactivation time constants of currents produced by rapid Ca(2+) concentration jumps were voltage sensitive, but that the activation time constants were Ca(2+) sensitive. The deactivation time constants increased linearly with the log of membrane potential. The qualitatively different behaviors of this channel in response to different Ca(2+) concentrations adds a new dimension to Ca(2+) signaling: the same channel can mediate either excitatory or inhibitory responses, depending on the amplitude of the cellular Ca(2+) signal.

Show MeSH
Related in: MedlinePlus