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

Non-technicalsummary 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 trans- verse tubular system, and the sarcoplasmic reticulum (SR) calcium release channel (so-called ryanodine receptor channel). This paper demonstrates that adult skeletal muscle fibres trans- fected 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. Abstract We investigated the effects of the overexpression of two enhanced green fluorescent protein (EGFP)-tagged α1sDHPR variants on Ca 2+ currents (ICa), charge movements (Q )a nd SR Ca 2+ release of musclefibres isolated from adult mice. Flexor digitorum brevis (FDB) muscles were transfected by in vivo electroporation with plasmids encoding for EGFP-α1sDHPR-wt and EGFP-α1sDHPR-T935Y (an isradipine-insensitive mutant). Two-photon laser scanning micro- scopy (TPLSM) was used to study the subcellular localization of transgenic proteins, while ICa, Q and Ca 2+ release were studied electrophysiologically and optically under voltage-clamp conditions. TPLSM images demonstrated that most of the transgenic α1sDHPR 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-α1sDHPR-wt in quantitiescomparabletothatofnative α1sDHPR.Thoughexpressionofbothtransgenicvariants ofthe alphasubunitof thedihydropyridinereceptor (α1sDHPR) resultedin ∼50%increaseinQ, they surprisingly had no effect on the maximal Ca 2+ conductance (gCa )n or the SR Ca 2+ release. Nonetheless,fibresexpressingEGFP-α1sDHPR-T935Yexhibitedupto70%isradipine-insensitive ICa (ICa-ins) with a right-shifted voltage dependence compared to that in control fibres. Inter- estingly,QandSRCa 2+ releasealsodisplayedright-shiftedvoltagedependenceinfibresexpressing EGFP-α1sDHPR-T935Y.In contrast, the midpointsof the voltage dependenceofgCa,Q and Ca 2+ releasewerenotdifferentfromthoseincontrolfibresandinfibresexpressingEGFP-α1sDHPR-wt. Overall, our results suggest that transgenic α1sDHPRs are correctly trafficked and inserted in the TTSmembrane,andthatasubstantialfractionofthemworksasconductiveCa 2+ channelscapable of physiologically controlling the release of Ca 2+ from the SR. A plausible corollary of this work is that the expression of transgenic variants of the α1sDHPR 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.