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Biological Research

Print version ISSN 0716-9760

Biol. Res. vol.37 no.4 Santiago  2004

http://dx.doi.org/10.4067/S0716-97602004000400015 

 

Biol Res 37: 609-612, 2004

ARTICLE

Regulation of cardiac excitation-contraction coupling by sorcin, a novel modulator of ryanodine receptors

EMILY F. FARRELL, ANAID ANTARAMIAN, NANCY BENKUSKY, XINSHENG ZHU, ANGÉLICA RUEDA, ANA M. GÓMEZ* and HÉCTOR H. VALDIVIA

Department of Physiology, University of Wisconsin Medical School, Madison, WI 53706, USA and *INSERM, Montepellier, France.

Dirección para Correspondencia


ABSTRACT

Activation of Ca2+ release channels/ryanodine receptors (RyR) by the inward Ca2+ current (ICa) gives rise to Ca2+-induced Ca2+ release (CICR), the amplifying Ca2+ signaling mechanism that triggers contraction of the heart. CICR, in theory, is a high-gain, self-regenerating process, but an unidentified mechanism stabilizes it in vivo. Sorcin, a 21.6 kDa Ca2+-binding protein, binds to cardiac RyRs with high affinity and completely inhibits channel activity. Sorcin significantly inhibits both the spontaneous activity of RyRs in quiescent cells (visualized as Ca2+ sparks) and the ICa-triggered activity of RyRs that gives rise to [Ca2+]i transients. Since sorcin decreases the amplitude of the [Ca2+]i transient without affecting the amplitude of ICa, the overall effect of sorcin is to reduce the "gain" of excitation-contraction coupling. Immunocytochemical staining shows that sorcin localizes to the dyadic space of ventricular cardiac myocytes. Ca2+ induces conformational changes and promotes translocation of sorcin between soluble and membranous compartments, but the [Ca2+] required for the latter process (ED50 = ~200 mM) appears to be reached only within the dyadic space. Thus, sorcin is a potent inhibitor of both spontaneous and ICa-triggered RyR activity and may play a role in helping terminate the positive feedback loop of CICR.

Key words: Sorcin, ryanodine receptors, CICR, dihydropyridine receptor, sarcoplasmic reticulum.


Excitation-contraction (E-C) coupling in cardiac myocytes is the process by which depolarization of the cell, by way of action potentials, leads to contraction. DHPRs, localized to transverse-tubule (T-tubule) membranes open in response to the depolarization to allow an influx of extracellular Ca2+. This incoming Ca2+ ("trigger Ca2+") binds to ryanodine receptors (RyRs) of the SR (see Franzini-Armstrong, this issue) and causes Ca2+ release, giving rise to the process known as Ca2+-induced Ca2+ release (CICR). The concentration of Ca2+ released from the SR is high enough to bind to myofilaments and induce full contractions.

E-C coupling must be tightly regulated in order to insure proper functioning of the heart. Thus, the RyR, one of the main determinants of E-C coupling, associates with several endogenous modulators and proteins, resulting in a versatile, flexible, and complex regulation of Ca2+ release. However, none of these accessory proteins is capable of causing complete RyR channel closure, a mechanism that is desirable to quench the inherently positive feedback of CICR. A novel protein, sorcin, has recently emerged as a powerful modulator of RyRs.

Sorcin (formerly V-19) is a 21.6 kDa protein originally discovered in multi-drug-resistant cells, where it was thought to have a role in drug resistance. However, it was later found that sorcin expression did not correlate with drug resistance, and thus its function remained incompletely understood.

Furthermore, the expression of sorcin in a wide variety of mammalian tissues including heart, skeletal muscle, brain and kidney, clearly indicates that its function transcends drug resistance.

Valuable information came about with the elucidation of sorcin's primary structure. Sorcin displays sequence similarity with calpain and grancalcin, two members of the penta-EF hand family of proteins. Sorcin binds Ca2+ with high affinity (KD,Ca ~1 mM), and Ca2+-bound sorcin undergoes conformational changes that allow it to translocate from soluble compartments (cytosol) to membranes, where presumably associates to target proteins. Thus, in the heart, where Ca2+ raises and falls at concentrations that effectively saturate sorcin, this protein has the capacity to associate and dissociate from its membrane-bound target on a beat-to-beat basis. But a role for sorcin in the heart (and indeed, in any other tissue) has not been definitively established.

Another breakthrough in the elucidation of the role of sorcin's function resulted from the work of Meyers, et al. (1995), who found that forced expression of sorcin in fibroblasts also resulted in expression of RyRs and that sorcin antibodies co-immunoprecipitated RyRs from cardiac myocytes. These results prompted investigation into sorcin's possible functional effects on the RyR, and in 1997, Lokuta et al. revealed through single channel studies and [3H]-ryanodine binding that sorcin is capable of completely inhibiting cardiac RyR with high affinity (Kd ­ 1 mM). This result encouraged further probing into sorcin's regulatory roles. Lokuta et al. (1997) also showed that Protein Kinase A (PKA) is able to diminish sorcin's inhibitory effects on RyRs. PKA's effects are interesting in that b-adrenergic stimulation, a mechanism that modulates E-C coupling during high metabolic demands or in the setting of exercise, activates PKA. Therefore, sorcin itself may be under the influence of b-adrenergic stimuli. Most importantly, sorcin's inhibitory effect on RyRs suggested that a possible role for this protein is the regulation of Ca2+ release during E-C coupling.

However, this proposed scheme of sorcin function may be more complicated. Sorcin has also been demonstrated to bind to the DHPR as well (Meyers 1998). The DHPR, as mentioned previously, is the voltage-gated Ca2+ channel of T-tubules that is activated when action potentials propagate along the cell membrane. Its role in E-C coupling of the heart is primordial, as it provides RyRs with Ca2+, the essential stimulus that triggers and grades Ca2+ release. Meyers et al. (1998) were able to co-immunoprecipitate the a1 pore-forming subunit of the DHPR with a sorcin antibody. Binding of sorcin was localized to the C-terminus of the a1 subunit, as C-terminus-truncated fragments of the DHPR could not be immunoprecipitated. Although the functional consequences of such interaction were not investigated, it became clear that sorcin is capable of binding to both, the RyR and the DHPR, at least in vitro. However, many questions remain unanswered: what is the effect of Sorcin on DHPRs? Is this effect complementary or antagonistic to that exerted on RyRs? Under what conditions does the binding of sorcin to a given channel predominate? What is the overall effect of sorcin on E-C coupling of an intact, fully functional cardiac cell? Can whole hearts perform well without sorcin? Are any of the cardiac effects of sorcin translatable to other cells that express it?

Figure 1. Hypothetical role for sorcin in ventricular cells. See text for details.

 

While work to solve the above questions is in progress, we propose the following hypothesis for sorcin's role in E-C coupling of the heart (see diagram below) based on the literature and on results obtained in our laboratory: during diastole, a portion of sorcin is attached to RyRs, although it is pertinent to consider that other protein targets may also bind sorcin at this [Ca2+] (for example, Meyers et al. (1998) showed that sorcin may bind to the carboxyl-end of DHPRs, but we have been unable to detect effects of this potential interaction). We postulate that the functional relevance of this membrane-bound sorcin during diastole is to prevent "Ca2+ leak" from RyRs. One assumption of this postulate is that the Ca2+ spark rate of quiescent cells should increase upon cell permeabilization, as this pool of sorcin eventually detaches from protein targets. As Ca2+ in the dyadic cleft rapidly reaches peak levels due to ICa and its associated CICR, sorcin undergoes drastic conformational changes due to Ca2+ association to at least two of its five EF-hands (Xie 2001; Mella 2003). This conformational change exposes hydrophobic residues and prompts sorcin to bind to membrane-bound protein targets by mechanisms probably similar to those of calpain and grancalcin, two other members of the penta EF-hand family of proteins. Sorcin translocation is induced and prompts sorcin's association to RyRs, which inhibits further Ca2+ release, quickly quenching CICR. As Ca2+ dissipates from the dyadic cleft, the Ca2+-sorcin interaction also fades, inducing sorcin to adopt its Ca2+-free conformational state, decreasing affinity for the RyR, and reversing sorcin's translocation back to cytosolic compartments. No further Ca2+ release should be elicited after sorcin dissociation of RyRs as the stimulating [Ca2+] quickly dissipates. When the dyadic cleft reverts to resting [Ca2+] during diastole, the entire process is now ready to repeat itself. A hole to fill in this hypothetical scenario is the effect of sorcin on DHPRs, which as mentioned above is unknown but could be complementary to that on RyRs (inhibition). This scenario is consistent with available data, but obviously, more investigation is required.

ACKNOWLEDGEMENTS

We wish to thank the National Institutes of Health and the American Heart Association for grant support.

REFERENCES

FRANZINI-ARMSTRONG C (2004) Functional implications of RyR-DHPR relationships in skeletal and cardiac muscles. Biol Res 37: 507-512         [ Links ]

LOKUTA AJ, MEYERS MB, FISHMAN G, SANDER PR, VALDIVIA HH (1997) Regulation of cardiac ryanodine receptors by sorcin. J Biol Chem 272:25333-25338         [ Links ]

MELLA M, COLOTTI G, ZAMPARELLI C, VERZILI D, ILARI A, CHIANCONE E (2003) Information transfer in the penta-EF-hand protein sorcin does not operate via the canonical structural/functional pairing: a study with site-specific mutants. J Biol Chem 278:24921-24928         [ Links ]

MEYERS MB, PICKEL VM, SHEU S-S, SHARMA VK, SCOTTO KW, FISHMAN GI (1995) Association of sorcin with the cardiac ryanodine receptor. J Biol Chem 270:26411-26418         [ Links ]

MEYERS MB, PURI TS, CHIEN AJ, GAO T, HSU PH, HOSEY MM, FISHMAN GI (1998) Sorcin associates with the pore-forming subunit of voltage-dependent L-type Ca2+ channels. J Biol Chem 273:18930-18935         [ Links ]

XIE X, DWYER MD, SWENSON L, PARKER MH, BOTFIELD MC (2001) Crystal structure of calcium-free human sorcin: a member of the penta-EF-hand protein family. Protein Sci 10:2419-2425         [ Links ]

 

Corresponding author: Héctor H. Valdivia, MD, PhD., University of Wisconsin Medical School, 1300 University Ave., Madison, WI 53706, USA. Telephone: (608) 265-5960, Fax: (608) 265-5512, E-mail: valdivia@physiology.wisc.edu

Received: March 11, 2004. Accepted: April 15, 2004.

 

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