Abstract
Sorcin is a widely expressed, 22-kDa Ca2+-binding protein initially identified in multidrug-resistant cells. In the heart, sorcin localizes to the dyadic junctions of transverse tubules and sarcoplasmic reticulum and coimmunoprecipitates with the Ca2+ release channel/ryanodine receptor (RyR) (Meyers, M. B., Pickel, V. M., Sheu, S.-S., Sharma, V. K., Scotto, K. W., and Fishman, G. I. (1995) J. Biol. Chem. 270, 26411-26418). We have investigated a possible functional interaction between sorcin and cardiac RyR using purified recombinant sorcin in [3H]ryanodine binding experiments and single channel recordings of RyR. The open probability of single RyR was decreased significantly by the addition of sorcin to the cytoplasmic side of the channel (IC50 approximately 480 nM). In addition, sorcin completely inhibited [3H]ryanodine binding with an IC50 approximately 700 nM. Inhibition occurred over a wide range of [Ca2+], and sorcin-modulated RyR remained Ca2+-dependent. Furthermore, caffeine-activated RyRs were also inhibited by sorcin at low [Ca2+] (pCa 7), suggesting that Ca2+ is not an obligatory factor for sorcin inhibition of RyR. Comparisons of these inhibitory effects with those of calmodulin and calpain, proteins structurally related to sorcin, suggested that the interaction of sorcin with cardiac RyR was distinct from and independent of either of these modulatory proteins. Phosphorylation of sorcin with the catalytic subunit of protein kinase A significantly decreased the ability of sorcin to modulate RyR. These results suggest that sorcin may modulate RyR function in a normal cell environment and that the level of modulation is in turn influenced by signaling pathways that increase protein kinase A activity.
Highlights
Ca2ϩ-induced Ca2ϩ release (CICR),1 the process by which a small influx of extracellular Ca2ϩ triggers massive release of
We found that sorcin inhibits ryanodine receptor (RyR) activity in a dose-dependent manner via a mechanism different from that exerted by calmodulin or calpain
To investigate if sorcin remained associated with the sarcoplasmic reticulum (SR)-enriched microsomes used in these experiments, we carried out Western blot analysis of total cardiac homogenate and of SR microsomes
Summary
A variety of endogenous substances regulate the activity of RyR, including Ca2ϩ (which is the primary signal for Ca2ϩ release), Mg2ϩ, ATP, Hϩ, calmodulin, and several protein kinases [6, 7] Exogenous substances such as ryanodine [8], caffeine [9], and scorpion peptides [10], without a role in CICR, regulate RyRs and have contributed to define their pharmacological profile. A role for sorcin in multidrug resistance has not been elucidated, the protein may have a normal function as an accessory protein of RyR/ Ca2ϩ release channels. To explore the potential functional consequences of a RyRsorcin interaction and its implications for CICR in the heart, we conducted [3H]ryanodine binding experiments and single channel recordings of cardiac RyR and tested the effect of recombinant sorcin.
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