Search

It is well-established that in heart, both the L-type Ca2+ channel and the cystic fibrosis transmembrane conductance regulator Cl- channel are regulated by cAMP-dependent phosphorylation. However, it is not clear whether both of these channels are regulated in concert by protein kinase A (PKA) or whether there are mechanisms that independently control the phosphorylation of these two PKA targets. The purpose of this study was to compare the effects of various protein phosphatase and protein kinase inhibitors on these two ionic currents (ICa and ICl) in guinea pig ventricular myocytes to gain insight into these questions. We found that both the stimulation and washout of the effects of isoproterenol on ICl are about twice as fast as the effects on ICa, probably because the dephosphorylation reaction for ICl is faster than that for ICa. In contrast, inhibition of protein phosphatases with 10 microM microcystin stimulated both ICa and ICl, but the stimulation of ICl was much slower and smaller than the stimulation of ICa. The effect of microcystin was inhibited by staurosporine (Ki = 171.5 and 161 nM for ICa and ICl, respectively), suggesting that the stimulation was due to a kinase. The kinase was not protein kinase C (PKC) because it was not inhibited by the specific pseudosubstrate inhibitor of PKC, PKC(19-31), and it was not PKA because it was not inhibited by adenosine 3',5'-cyclic phosphorothioate. These results suggest that although both the Ca2+ and Cl- channels are regulated by cAMP-dependent phosphorylation, another protein kinase may also regulate these channels, and the kinetics of the response of the channels to phosphorylation can be modulated independently by protein phosphatases.

Adenophostin-A, a novel compound isolated from cultures of Penicillium brevicompactum, has been shown to stimulate Ca2+ release from inositol-1,4,5-trisphosphate (IP3)-sensitive Ca2+ stores in microsomal preparations, permeabilized cells, and lipid vesicles containing purified IP3 receptor. The purpose of the current study was to compare the effects of adenophostin-A and IP3 on Ca2+ release from stores and Ca2+ influx in intact Xenopus laevis oocytes. Ca2+ influx though store-operated Ca2+ channels and Ca2+ release from stores were monitored by measuring two Ca2+ -activated Cl- currents that can be used as real-time indicators of Ca2+ release and Ca2+ influx (I(Cl-1) and I(Cl-2), respectively). We find that high concentrations (final intraoocyte concentrations of 5-10 microM) of adenophostin-A and IP3 stimulate a large Ca2+ release from stores (as measured by I(Cl-1)) followed by Ca2+ influx (as measured by I(Cl-2)). Low concentrations (approximately 50 nM) of IP3 stimulate oscillations in Ca2+ release without stimulating Ca2+ influx. In contrast, low concentrations of adenophostin-A can stimulate Ca2+ influx without stimulating a large Ca2+ release. However, Ca2+ influx did not occur in the complete absence of Ca2+ release. Therefore, it is unlikely that adenophostin-A directly stimulates store-operated Ca2+ channels. We hypothesize that adenophostin-A releases Ca2+ from a subpopulation of stores that is tightly coupled to store-operated Ca2+ channels.