Ca2+-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 Ca2+ and voltage. Excised inside-out patches containing many Ca2+-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 [Ca2+]. However, depending on the amplitude of the Ca2+ signal, this channel exhibited qualitatively different behaviors. At [Ca2+] < 1 μM, the currents activated slowly upon depolarization and deactivated upon hyperpolarization and the steady state current–voltage relationship was strongly outwardly rectifying. At higher [Ca2+], the currents did not rectify and were time independent. This difference in behavior at different [Ca2+] was explained by an apparent voltage-dependent Ca2+ sensitivity of the channel. At +120 mV, the EC50 for channel activation by Ca2+ was approximately fourfold less than at −120 mV (0.9 vs. 4 μM). Thus, at [Ca2+] < 1 μM, inward current was smaller than outward current and the currents were time dependent as a consequence of voltage-dependent changes in Ca2+ binding. The voltage-dependent Ca2+ sensitivity was explained by a kinetic gating scheme in which channel activation was Ca2+ dependent and channel closing was voltage sensitive. This scheme was supported by the observation that deactivation time constants of currents produced by rapid Ca2+ concentration jumps were voltage sensitive, but that the activation time constants were Ca2+ sensitive. The deactivation time constants increased linearly with the log of membrane potential. The qualitatively different behaviors of this channel in response to different Ca2+ concentrations adds a new dimension to Ca2+ signaling: the same channel can mediate either excitatory or inhibitory responses, depending on the amplitude of the cellular Ca2+ signal.

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Ca-activated Cl currents are widely expressed in many cell types and play diverse and important physiological roles. The Xenopus oocyte is a good model system for studying the regulation of these currents. We previously showed that inositol 1,4,5-trisphosphate (IP3) injection into Xenopus oocytes rapidly elicits a noninactivating outward Cl current ( I Cl1-S) followed several minutes later by the development of slow inward ( I Cl2) and transient outward ( I Cl1-T) Cl currents. In this paper, we investigate whether these three currents are mediated by the same or different Cl channels. Outward Cl currents were more sensitive to Ca than inward Cl currents, as shown by injection of different amounts of Ca or by Ca influx through a heterologously expressed ligand-gated Ca channel, the ionotropic glutamate receptor iGluR3. These data could be explained by two channels with different Ca affinities or one channel with a higher Ca affinity at depolarized potentials. To distinguish between these possibilities, we determined the anion selectivity of the three currents. The anion selectivity sequences for the three currents were the same (I > Br > Cl), but I Cl1-Shad an I-to-Cl permeability ratio more than twofold smaller than the other two currents. The different anion selectivities and instantaneous current-voltage relationships were consistent with at least two different channels mediating these currents. However, after consideration of possible errors, the hypothesis that a single type of Cl channel underlies the complex waveforms of the three different macroscopic Ca-activated Cl currents in Xenopus oocytes remains a viable alternative.

Triadin, a calsequestrin-anchoring transmembrane protein of the sarcoplasmic reticulum (SR), was successfully purified from the heavy fraction of SR (HSR) of rabbit skeletal muscle with an anti-triadin immunoaffinity column. Since depletion of triadin from solubilized HSR with the column increased the [3H]ryanodine binding activity, we tested a possibility of triadin for a negative regulator of the ryanodine receptor/Ca2+ release channel (RyR). Purified triadin not only inhibited [3H]ryanodine binding to the solubilized HSR but also reduced openings of purified RyR incorporated into the planar lipid bilayers. On the other hand, calsequestrin, an endogenous activator of RyR [Kawasaki and Kasai (1994) Biochem. Biophys. Res. Commun. 199, 1120-1127; Ohkura et al. (1995) Can. J. Physiol. Pharmacol. 73, 1181-1185] potentiated [3H]ryanodine binding to the solubilized HSR. Ca2+ dependency of [3H]ryanodine binding to the solubilized HSR was reduced by triadin, whereas that was enhanced by calsequestrin. Interestingly, [3H]ryanodine binding to the solubilized HSR potentiated by calsequestrin was reduced by triadin. Immunostaining with anti-triadin antibody proved that calsequestrin inhibited the formation of oligomeric structure of triadin. These results suggest that triadin inhibits the RyR activity and that RyR is regulated by both triadin and calsequestrin, probably through an interaction between them. In this paper, triadin has been first demonstrated to have an inhibitory role in the regulatory mechanism of the RyR.