Excitation-contraction (EC) coupling in skeletal muscle depends upon trafficking of Ca V 1.1, the principal subunit of the dihydropyridine receptor (DHPR) (L-type Ca 2+ channel), to plasma membrane regions at which the DHPRs interact with type 1 ryanodine receptors (RyR1) in the sarcoplasmic reticulum. A distinctive feature of this trafficking is that Ca V 1.1 expresses poorly or not at all in mammalian cells that are not of muscle origin (e.g., tsA201 cells), in which all of the other nine Ca V isoforms have been successfully expressed. Here, we tested whether plasma membrane trafficking of Ca V 1.1 in tsA201 cells is promoted by the adapter protein Stac3, because recent work has shown that genetic deletion of Stac3 in skeletal muscle causes the loss of EC coupling. Using fluorescently tagged constructs, we found that Stac3 and Ca V 1.1 traffic together to the tsA201 plasma membrane, whereas Ca V 1.1 is retained intracellularly when Stac3 is absent. Moreover, L-type Ca 2+ channel function in tsA201 cells coexpressing Stac3 and Ca V 1.1 is quantitatively similar to that in myotubes, despite the absence of RyR1. Although Stac3 is not required for surface expression of Ca V 1.2, the principle subunit of the cardiac/brain L-type Ca 2+ channel, Stac3 does bind to Ca V 1.2 and, as a result, greatly slows the rate of current inactivation, with Stac2 acting similarly. Overall, these results indicate that Stac3 is an essential chaperone of Ca V 1.1 in skeletal muscle and that in the brain, Stac2 and Stac3 may significantly modulate Ca V 1.2 function.Stac adaptor protein | L-type Ca 2+ channel | excitation-contraction coupling

In skeletal muscle, conformational coupling between Ca V 1.1 in the plasma membrane and type 1 ryanodine receptor (RyR1) in the sarcoplasmic reticulum (SR) is thought to underlie both excitation-contraction (EC) coupling Ca 2+ release from the SR and retrograde coupling by which RyR1 increases the magnitude of the Ca 2+ current via Ca V 1.1. Recent work has shown that EC coupling fails in muscle from mice and fish null for the protein Stac3 (SH3 and cysteine-rich domain 3) but did not establish the functional role of Stac3 in the Ca V 1.1-RyR1 interaction. We investigated this using both tsA201 cells and Stac3 KO myotubes. While confirming in tsA201 cells that Stac3 could support surface expression of Ca V 1.1 (coexpressed with its auxiliary β 1a and α 2 -δ 1 subunits) and the generation of large Ca 2+ currents, we found that without Stac3 the auxiliary γ 1 subunit also supported membrane expression of Ca V 1.1/β 1a /α 2 -δ 1 , but that this combination generated only tiny Ca 2+ currents. In Stac3 KO myotubes, there was reduced, but still substantial Ca V 1.1 in the plasma membrane. However, the Ca V 1.1 remaining in Stac3 KO myotubes did not generate appreciable Ca 2+ currents or EC coupling Ca 2+ release. Expression of WT Stac3 in Stac3 KO myotubes fully restored Ca 2+ currents and EC coupling Ca 2+ release, whereas expression of Stac3 W280S (containing the Native American myopathy mutation) partially restored Ca 2+ currents but only marginally restored EC coupling. We conclude that membrane trafficking of Ca V 1.1 is facilitated by, but does not require, Stac3, and that Stac3 is directly involved in conformational coupling between Ca V 1.1 and RyR1.L-type Ca 2+ channels | Stac3 protein | excitation-contraction coupling

In skeletal muscle, excitation–contraction coupling between CaV1.1 and RyR1 depends on the presence of a critical domain (residues 720–764/5) within the cytoplasmic II–III loop of CaV1.1. Polster et al. identify the adaptor protein Stac3 as a direct interaction partner of the critical domain.

The proximal C terminus of the cardiac L-type calcium channel (Ca V 1.2) contains structural elements important for the binding of calmodulin (CaM) and calcium-dependent inactivation, and exhibits extensive sequence conservation with the corresponding region of the skeletal L-type channel (Ca V 1.1). However, there are several Ca V 1.1 residues that are both identical in six species and are non-conservatively changed from the corresponding Ca V 1.2 residues, including three of the "IQ motif." To investigate the functional significance of these residue differences, we used native gel electrophoresis and expression in intact myotubes to compare the binding of CaM to extended regions (up to 300 residues) of the C termini of Ca V 1.1 and Ca V 1.2. We found that in the presence of Ca 2؉ (either millimolar or that in resting myotubes), CaM bound strongly to C termini of Ca V 1.2 but not of Ca V 1.1. Furthermore, replacement of two residues (Tyr 1657 and Lys 1662 ) within the IQ motif of a C-terminal Ca V 1.2 construct with the divergent residues of Ca V 1.1 (His 1532 and Met 1537 ) led to a weakening of CaM binding (native gels), whereas the reciprocal substitution in Ca V 1.1 caused a gain of CaM binding. In full-length Ca V 1.2, substitution of these same two divergent residues with those of Ca V 1.1 (Y1657H, K1662M) eliminated calcium-dependent inactivation of the heterologously expressed channel. Thus, our results reveal that a conserved difference between the IQ motifs of Ca V 1.2 and Ca V 1.1 has a profound effect on both CaM binding and calciumdependent inactivation.

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