STAC Proteins Associate to the IQ Domain of CaV1.2 and Inhibit Calcium-Dependent Inactivation

Abstract

The adaptor proteins STAC1, STAC2, and STAC3 represent a newly identified family of regulators of voltage-gated calcium channel (Ca V ) trafficking and function. The skeletal muscle isoform STAC3 is essential for excitation-contraction coupling and its mutation causes severe muscle disease. Recently, two distinct molecular domains in STAC3 were identified, necessary for its functional interaction with Ca V 1.1: the C1 domain, which recruits STAC proteins to the calcium channel complex in skeletal muscle triads, and the SH3-1 domain, involved in excitation-contraction coupling. These interaction sites are conserved in the three STAC proteins. However, the molecular domain in Ca V 1 channels interacting with the STAC C1 domain and the possible role of this interaction in neuronal Ca V 1 channels remained unknown. Using Ca V 1.2/2.1 chimeras expressed in dysgenic (Ca V 1.1) myotubes, we identified the amino acids 1,641-1,668 in the C terminus of Ca V 1.2 as necessary for association of STAC proteins. This sequence contains the IQ domain and alanine mutagenesis revealed that the amino acids important for STAC association overlap with those making contacts with the C-lobe of calcium-calmodulin (Ca/CaM) and mediating calcium-dependent inactivation of Ca V 1.2. Indeed, patch-clamp analysis demonstrated that coexpression of either one of the three STAC proteins with Ca V 1.2 opposed calcium-dependent inactivation, although to different degrees, and that substitution of the Ca V 1.2 IQ domain with that of Ca V 2.1, which does not interact with STAC, abolished this effect. These results suggest that STAC proteins associate with the Ca V 1.2 C terminus at the IQ domain and thus inhibit calcium-dependent feedback regulation of Ca V 1.2 currents.ecently STAC3 (SH3 and cysteine-rich containing protein 3) has been identified as an essential regulator of calcium channel trafficking and function in skeletal muscle excitationcontraction (EC) coupling and a mutation in STAC3 has been linked to the severe muscle disease Native American myopathy (NAM) (1, 2). Expression of STAC3 is restricted to skeletal muscle, where STAC3 associates with the voltage-gated calcium channel Ca V 1.1 and is involved in mediating voltage-induced calcium release from the sarcoplasmic reticulum (3, 4). In nonmuscle cells, STAC3 was shown to facilitate functional membrane expression of Ca V 1.1 and alter the current properties of Ca V 1.2 (5), suggesting a role of STAC proteins as an L-type calcium channel (Ca V 1) regulator. STAC3 belongs to a family of adaptor proteins that comprises two additional isoforms, STAC1 and STAC2, which are highly expressed in the brain (1).All three STAC proteins contain one C1 domain and two SH3 protein interaction domains (6). We previously demonstrated that the stable association of STAC3 to the Ca V 1 channel complex in the triads of skeletal muscle relies on the C1 domain (7). In contrast, the NAM mutation, which impairs EC coupling and is located in the SH3-1 domain of STAC3 (2-4), did not abolish the interaction with ...

Crystal structures of Ca 2+ –calmodulin bound to Na V C-terminal regions suggest role for EF-hand domain in binding and inactivation

“…Given the similarities between Na V and Ca V channels in regard to CDI (21), similar mechanisms may exist for several isoforms of Ca V s, whereby the position of the EF-hand domain regulates CDI. Interestingly, STAC proteins have recently been found to engage Ca V s (51)(52)(53)(54)(55)(56)(57) and were shown to interact with the EF-hand domain, abolishing CDI (26). This is reminiscent of the effect of FHFs on Na V s, and it was shown that FHFs can abolish CDI in Na V 1.4 (26).…”

“…Interestingly, FHFs have been shown to abolish CDI in Na V 1.4 (26), an effect that may be ascribed to its inherent effect on the EF-hand orientation, locking it into a position such that no Ca 2+ -dependent conformational change occurs. Similarly, STAC proteins, which can bind to two different regions in Ca V 1 channels (26,(51)(52)(53)(54)(55)(56), including the EFhand domain (26), could affect the relative orientation between EF-hand and IQ domain. How this conformational change further results in inactivation remains to be shown, but of note is the ability of the EF-hand domain to interact with the III-IV linker, and that this interaction affects channel inactivation (14).…”

Presynaptic α2δ subunits are key organizers of glutamatergic synapses

“…Electron microscopy, structural analysis: Cultures of neurons were prepared as described above with the exception, that neurons were grown on coverglasses coated with a carbon layer as previously described (Campiglio et al, 2018), and fixed with 2% glutaraldehyde (Agar Scientific Ltd., Stansted, UK) in phosphate buffer (PB; 0.1 M, pH 7.4). After washing in PB three times 5 min at RT, neurons were post-fixed and stained with 0.2% osmium tetroxide (Electron Microscopy Sciences, Hatfield, PA) in PB (w/v) for 30 min at RT.…”

Excitation-contraction coupling in skeletal muscle: recent progress and unanswered questions

“…Several years ago, Polster et al (2015) demonstrated that STAC3 facilitates expression and membrane targeting of Ca V 1.1 in tsA201 cells, and later, this function of STAC3 was shown to be due to the interaction of the PKC C1 domain of STAC3 with the C-terminal domain of the DHPR-α 1s (Campiglio et al 2018a;Campiglio and Flucher 2017). This interaction was also shown to play a role in the inhibition of calcium-dependent inactivation of Ca V channels (Campiglio et al 2018a;Niu et al 2018a;Niu et al 2018b); however, it does not appear to be directly relevant to the ECC.…”