Functional expression of transgenic α1sDHPR channels in adult mammalian skeletal muscle fibres

Non-technical summary In mammalian skeletal muscle, the coupling between action potential activation and contraction is supposed to be ultimately mediated by the interaction of two ion channels, the L-type calcium channel (so-called dihydropyridine receptor channel) at the transverse tubular system, and the sarcoplasmic reticulum (SR) calcium release channel (so-called ryanodine receptor channel). This paper demonstrates that adult skeletal muscle fibres transfected in vivo with DNA plasmids are able to express functional transgenic dihydropyridine receptor channels. More importantly, the data suggest that transgenic dihydropyridine receptor channels replace native channels in their interaction with SR calcium release channels. Our findings open new avenues for structural and functional studies of the molecular interactions underlying excitation-contraction coupling within the physiologically relevant cellular context of adult mammalian skeletal muscle fibres.We investigated the effects of the overexpression of two enhanced green fluorescent protein (EGFP)-tagged alpha 1sDHPR variants on Ca2+ currents (I-Ca), charge movements (Q) and SR Ca2+ release of muscle fibres isolated from adult mice. Flexor digitorum brevis (FDB) muscles were transfected by in vivo electroporation with plasmids encoding for EGFP-alpha 1sDHPR-wt and EGFP-alpha 1sDHPR-T935Y (an isradipine-insensitive mutant). Two-photon laser scanning microscopy (TPLSM) was used to study the subcellular localization of transgenic proteins, while I-Ca, Q and Ca2+ release were studied electrophysiologically and optically under voltage-clamp conditions. TPLSM images demonstrated that most of the transgenic alpha 1sDHPR was correctly targeted to the transverse tubular system (TTS). Immunoblotting analysis of crude extracts of transfected fibres demonstrated the synthesis of bona fide transgenic EGFP-alpha 1sDHPR-wt in quantities comparable to that of native alpha 1sDHPR. Though expression of both transgenic variants of the alpha subunit of the dihydropyridine receptor (alpha 1sDHPR) resulted in similar to 50% increase in Q, they surprisingly had no effect on the maximal Ca2+ conductance (g(Ca)) nor the SR Ca2+ release. Nonetheless, fibres expressing EGFP-alpha 1sDHPR-T935Y exhibited up to 70% isradipine-insensitive I-Ca (I-Ca-ins) with a right-shifted voltage dependence compared to that in control fibres. Interestingly, Q and SR Ca2+ release also displayed right-shifted voltage dependence in fibres expressing EGFP-alpha 1sDHPR-T935Y. In contrast, the midpoints of the voltage dependence of g(Ca), Q and Ca2+ release were not different from those in control fibres and in fibres expressing EGFP-alpha 1sDHPR-wt. Overall, our results suggest that transgenic alpha 1sDHPRs are correctly trafficked and inserted in the TTS membrane, and that a substantial fraction of them works as conductive Ca2+ channels capable of physiologically controlling the release of Ca2+ from the SR. A plausible corollary of this work is that the expression of transgenic variants of the alpha 1sDHPR leads to the replacement of native channels interacting with the ryanodine receptor 1 (RyR1), thus demonstrating the feasibility of molecular remodelling of the triads in adult skeletal muscle fibres.