Orai3 channel is the 2-APB-induced endoplasmic reticulum calcium leak.

We have studied in HeLa cells the molecular nature of the 2-APB induced ER Ca2+ leak using synthetic Ca2+ indicators that report changes in both the cytoplasmic ([Ca2+]i) and the luminal ER ([Ca2+]ER) Ca2+ concentrations. We have tested the hypothesis that Orai channels participate in the 2-APB-induced ER Ca2+ leak that was characterized in the companion paper. The expression of the dominant negative Orai1 E106A mutant, which has been reported to block the activity of all three types of Orai channels, inhibited the effect of 2-APB on the [Ca2+]ER but did not decrease the ER Ca2+ leak after thapsigargin (TG). Orai3 channel, but neither Orai1 nor Orai2, colocalizes with expressed IP3R and only Orai3 channel supported the 2-APB-induced ER Ca2+ leak, while Orai1 and Orai2 inhibited this type of ER Ca2+ leak. Decreasing the expression of Orai3 inhibited the 2-APB-induced ER Ca2+ leak but did not modify the ER Ca2+ leak revealed by inhibition of SERCA pumps with TG. However, reducing the expression of Orai3 channel resulted in larger [Ca2+]i response after TG but only when the ER store had been overloaded with Ca2+ by eliminating the acidic internal Ca2+ store with bafilomycin. These data suggest that Orai3 channel does not participate in the TG-revealed ER Ca2+ leak but forms an ER Ca2+ leak channel that is limiting the overloading with Ca2+ of the ER store.

Posttranscriptional regulation of the ß2-subunit of cardiac L-type Ca2+ channels by MicroRNAs during long-term exposure to isoproterenol in rats.

INTRODUCTION AND METHODS: The effects of long-term ß-adrenergic administration on the expression levels of the cardiac L-type Ca channel ß2 subunit, which regulates channel trafficking and function, were characterized in adult rats. RESULTS: Systemic administration of isoproterenol (150 mg·kg·h) for 2 d led to a 50% increase in the ventricular wet weight-to-body weight ratio (mg/g) and of more than two-fold in the expression of actin protein. In contrast, ß2 subunit protein levels decreased (down to 49%), while mRNA levels remained unchanged. Furthermore, levels of microRNAs (miRs), including miR-21 and miR-132, were upregulated (7.2 and 7.9 fold, respectively). Transfection of these miRs into HEK293 cells attenuated expression of a luciferase reporter gene controlled by a conserved 3'-untranslated region (UTR) of the ß2 subunit (down to 67% and 56%, respectively). Systemic administration of isoproterenol also led to briefer intracellular Ca transients during action potentials measured in isolated cardiomyocytes (down to 65%). CONCLUSION: These results suggest that cardiac L-type Ca channel ß2 subunit protein expression may be downregulated by miRs in response to long-term activation of ß-adrenergic signaling, possibly as an adaptive response in cardiac hypertrophy and sustained ß-adrenergic states.

Short-term regulation of excitation-contraction coupling by the beta1a subunit in adult mouse skeletal muscle.

The beta1a subunit of the skeletal muscle voltage-gated Ca2+ channel plays a fundamental role in the targeting of the channel to the tubular system as well as in channel function. To determine whether this cytosolic auxiliary subunit is also a regulatory protein of Ca2+ release from the sarcoplasmic reticulum in vivo, we pressure-injected the beta1a subunit into intact adult mouse muscle fibers and recorded, with Fluo-3 AM, the intracellular Ca2+ signal induced by the action potential. We found that the beta1a subunit significantly increased, within minutes, the amplitude of Ca2+ release without major changes in its time course. beta1a subunits with the carboxy-terminus region deleted did not show an effect on Ca2+ release. The possibility that potentiation of Ca2+ release is due to a direct interaction between the beta1a subunit and the ryanodine receptor was ruled out by bilayer experiments of RyR1 single-channel currents and also by Ca2+ flux experiments. Our data suggest that the beta1a subunit is capable of regulating E-C coupling in the short term and that the integrity of the carboxy-terminus region is essential for its modulatory effect.

AKAP79 increases the functional expression of skeletal muscle Ca2+ channels in Xenopus oocytes.

The actions of the kinase A anchoring protein, AKAP79, a key element in the regulation of the cardiac L-type Ca2+ channel, were assessed on skeletal muscle Ca2+ channels expressed in Xenopus oocytes. The channels were reconstituted by expressing the pore forming alpha1s subunit and its accessory subunits, alpha2-delta, beta, and gamma. We report, for the first time, that peak Ca2+ channel currents are greatly increased (3.5-fold) by AKAP79 when co-expressed with the truncated form of the alpha1s subunit. Immunoblots revealed that the increase in current amplitude is not accompanied by a corresponding increase in the membrane levels of the alpha1s subunit. This suggests that AKAP79 does not increase the trafficking of the channel. In addition, we show that the transcript of AKAP150, the rat ortholog of the human AKAP79, is expressed in rat skeletal muscle and propose that AKAP79/150 modulates Ca2+ channel function.