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Calmodulin and Excitation-Contraction Coupling

Susan L. Hamilton, Irina Serysheva and Gale M. Strasburg

S. Hamilton is a Professor and I. Serysheva is an Assistant Professor in the Department of Molecular Physiology and Biophysics at Baylor College of Medicine, 1 Baylor Plaza, Houston, TX 77030. G. M. Strasburg is a Professor in the Department of Food Science and Human Nutrition, Michigan State University, East Lansing, MI 48824-1224.

	Abstract

 
Excitation-contraction coupling in cardiac and skeletal muscle involves the transverse-tubule voltage-dependent Ca²⁺ channel and the sarcoplasmic reticulum Ca²⁺ release channel. Both of these ion channels bind and are modulated by calmodulin in both its Ca²⁺-bound and Ca²⁺-free forms. Calmodulin is, therefore, potentially an important regulator of excitation-contraction coupling. Its precise role, however, has not yet been defined.

	Introduction

Top Introduction CaM as an intracellular... CaM and the voltage-dependent... CaM and RYR1 A role for CaM... References

 
Excitation-contraction (E-C) coupling is the process by which depolarization of the muscle fiber membrane, elicited by a nerve action potential, triggers the release of Ca²⁺ from the sarcoplasmic reticulum (SR) (1). The resulting rise in intracellular Ca²⁺ concentration activates the troponin complex, thereby initiating the contraction of the muscle. The two primary proteins involved in the initial events of E-C coupling are the dihydropyridine receptor (DHPR) and the ryanodine receptor (RYR), which are both Ca²⁺ channels (Fig. 1). Skeletal and cardiac muscle have different isoforms of both the DHPR and RYR. The skeletal muscle DHPR, which is an L-type Ca²⁺ channel, is composed of four subunits: _1S (190–212 kDa), _2– (125 kDa), ß (52–58 kDa), and (25 kDa). The cardiac DHPR has three known subunits: _1C (240 kDa), _2– (125 kDa), and ß (62 kDa). The -subunit has not yet been identified as a subunit of the cardiac channel. The ₁-subunit of the DHPR forms the channel pore and contains the binding sites for channel-specific drugs.

View larger version (90K): [in this window] [in a new window]   FIGURE 1. Proteins involved in excitation-contraction coupling. Both the dihydropyridine receptor (DHPR) and skeletal muscle ryanodine receptor (RYR1) bind and are modulated by calmodulin (CaM). SR, sarcoplasmic reticulum; t-tubule, transverse-tubule. Greek letters represent DHPR subunits.

Release of Ca²⁺ from the SR is controlled by the Ca²⁺ release channel or RYR. The RYR1 and RYR2 are both homotetramers with a subunit molecular mass of ~565 kDa, and they share 66% sequence identity and ~80% overall homology. Approximately 4/5 of the RYRs are predicted to be cytoplasmic, with only 1/5 of the molecule at the carboxy terminus forming the luminal and membrane-spanning domains.

In addition to the primary mechanisms of regulation of E-C coupling (mechanical gating or CICR), Ca²⁺ release is likely to be modulated by other proteins bound to the DHPR or to RYR. One of these proteins, calmodulin (CaM), regulates the activity of the DHPR (8, 15), RYR1 (11), and RYR2 (13). On the basis of its interactions with these channels, CaM is likely to play an important role in both cardiac and skeletal muscle E-C coupling.

	CaM as an intracellular Ca2+ sensor

Top Introduction CaM as an intracellular... CaM and the voltage-dependent... CaM and RYR1 A role for CaM... References

 
CaM is a ubiquitously expressed Ca²⁺ binding protein composed of an amino terminal and a carboxy terminal lobe connected by an eight-turn -helix. The structures of CaM in its Ca²⁺-free (apoCaM) (5) and Ca²⁺-bound (Ca²⁺CaM) forms (obtained from the Brookhaven Protein Databank website: http://cmm.info.nih.gov/modeling/pdb_at_a_glance.html) are shown in Fig. 2. CaM binds most target proteins in a Ca²⁺-dependent manner. Both the amino terminal and carboxy terminal lobes have two E-F hand Ca²⁺ binding sites that undergo Ca²⁺-dependent conformational changes that expose hydrophobic binding pockets, allowing binding to amphipathic -helical domains within the target proteins (9). Ca²⁺CaM undergoes another major change in conformation on binding to its target. The structure of CaM bound to an amphipathic -helix from myosin light chain kinase is shown in Fig. 3B (4).

View larger version (37K): [in this window] [in a new window]   FIGURE 2. The structure of free CaM (apoCaM) [A, shown by nuclear magnetic resonance (NMR)] and bound CaM (Ca2+CaM) (B, shown by X-ray diffraction). N, amino terminal; C, carboxy terminal.

View larger version (34K): [in this window] [in a new window]   FIGURE 3. The structure of CaM-peptide complexes. A: apoCaM (blue) bound to an IQ domain on myosin (red) (theoretical model). B: Ca2+CaM (blue) complexed with its binding domain from rabbit skeletal myosin light chain kinase (red) (NMR images).

	CaM and the voltage-dependent Ca2+ channel

Top Introduction CaM as an intracellular... CaM and the voltage-dependent... CaM and RYR1 A role for CaM... References

 
CaM is both a positive and negative regulator of the cardiac L-type Ca²⁺ channel. Elevations in intracellular Ca²⁺ concentration produce a conformational change in CaM, tethered to the channel, producing L-channel inactivation (8). Zuhlke et al. (15) have shown that CaM serves as a Ca²⁺ sensor for both positive and negative regulation of the cardiac L-type Ca²⁺ channel (8). A mutant CaM that cannot bind Ca²⁺ at any of the four Ca²⁺ binding sites blocks the effects of Ca²⁺CaM on the L-type Ca²⁺ channel, suggesting that both the Ca²⁺-free and Ca²⁺-bound forms of CaM can bind to this channel. This ability of Ca²⁺CaM to inhibit the channel appears to be mediated via its binding to an IQ motif in the cytoplasmic carboxy tail of the ₁-subunit. If the isoleucine of this motif is mutated to an alanine, the Ca²⁺-dependent inactivation is lost, and this unmasks a strong facilitation by CaM. If the isoleucine is converted, however, to a glutamate, both of the effects of CaM (inactivation and facilitation) are lost (15). These findings suggest that either apoCaM and Ca²⁺CaM are binding in the same region of the DHPR (probably with different determinants for binding) or that Ca²⁺CaM binding sites are allosterically regulated by the binding of apoCaM.

	CaM and RYR1

Top Introduction CaM as an intracellular... CaM and the voltage-dependent... CaM and RYR1 A role for CaM... References

 
Dual regulation by CaM is also seen with RYR1. RYR1 is the major CaM binding protein of SR membranes (11). Tripathy et al. (11) have shown that CaM bound to SR Ca²⁺ release channel (RYR1) at nanomolar Ca²⁺ concentrations activates the channel. In contrast, CaM bound to RYR1 at micromolar Ca²⁺ concentrations inhibits Ca²⁺ release channel activity. Our studies demonstrate the existence of a single CaM binding site per subunit of RYR1 at both high and low Ca²⁺ concentrations (6). The affinity of both apoCaM and Ca²⁺CaM for RYR1 is in the range of 5–50 nM (6, 14, 11). Wagenknecht and colleagues (12) have identified the binding site for CaM in the three-dimensional reconstructions of RYR1, and the approximate location on RYR1 is shown in Fig. 1.

RYR1 itself binds Ca²⁺ in the absence of CaM, creating a complicated picture of the response of this channel to Ca²⁺ in the presence of CaM. In the absence of CaM, Ca²⁺ in the 1–300 µM range activates RYR1, but at concentrations of >500 µM it is inhibitory. In the presence of CaM, a biphasic dependence of RYR1 activity on Ca²⁺ is still seen, except both activation and inhibition take place at lower Ca²⁺ concentrations. A mutant CaM that does not bind Ca²⁺ at any of the four binding sites is an activator of the channel at all Ca²⁺ concentrations (10). The results of Fruen et al. (2) suggest that, in contrast to its interaction with RYR1, apoCaM binds with much lower affinity to RYR2 than to RYR1, and it does not increase RYR2 activity.

The binding sites for both apoCaM and Ca²⁺CaM on RYR1 appear to be close to amino acids 3630–3637. Bound CaM (either Ca²⁺-bound or Ca²⁺-free) can protect this region from either proteolytic cleavage after amino acid 3630 and after amino acid 3637 or modification of cysteine 3635 by either oxidants or N-ethylmaleimide (7). Cysteine 3635 can form a disulfide bond with an unidentified cysteine on an adjacent subunit (6). CaM bound to RYR1 can prevent this oxidation-induced intersubunit cross-linking. Conversely, oxidation blocks CaM binding (14). One interpretation of these findings is that CaM binds at a site of intersubunit contact and, in doing so, could protect the channel from the effects of oxidants (Fig. 4), for example, during periods of oxidative stress associated with strenuous exercise.

View larger version (36K): [in this window] [in a new window]   FIGURE 4. CaM binds at a site of subunit-subunit (s-s) contact on RYR1. The CaM binding site on RYR1 contains a cysteine that can be disulfide bonded to a second cysteine on a neighboring subunit (left). Ca2+CaM can protect the site from oxidation, and oxidation can prevent the binding of calmodulin (right).

	A role for CaM in E-C coupling?

Top Introduction CaM as an intracellular... CaM and the voltage-dependent... CaM and RYR1 A role for CaM... References

In summary, it seems likely that CaM binding to the DHPR and RYR must, in some way, modulate E-C coupling in both cardiac and skeletal muscle. Its role may be to regulate the Ca²⁺-dependent enhancement and inhibition of activity or, possibly, to prevent or enhance coupling between the DHPR and RYR1. Since CaM plays a wide variety of roles in both development and cell function, it would be difficult to create a CaM knockout animal model to determine the functional role of CaM in regulation of either cardiac or skeletal muscle E-C coupling. Instead, the most likely approach will be to mutate its binding sites on RYR1 and/or the DHPR, express these mutated channels in cells that are deficient in one of these proteins, and assess the functional consequences. This approach will require the further identification of the molecular determinants on RYR and the DHPR involved in both apoCaM and Ca²⁺CaM binding. If the amino acids needed for apoCaM binding are different from those for Ca²⁺CaM binding, it may be possible to selectively destroy the binding of one form of CaM without greatly altering the interaction of the other form. This would allow the assessment of the functional contributions of the different forms of CaM.

	References

Top Introduction CaM as an intracellular... CaM and the voltage-dependent... CaM and RYR1 A role for CaM... References

 

1. Franzini-Armstrong C and Protasi F. Ryanodine receptors of striated muscles: a complex channel capable of multiple interactions. Physiol Rev 77: 699–729, 1997.[Abstract/Free Full Text]
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3. Houdusse A, Silver M, and Cohen C. A model of Ca²⁺-free calmodulin binding to unconventional myosins reveals how calmodulin acts as a regulatory switch. Structure 4: 1475–1490, 1996.[Medline]
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