In the DHPRβ1-null zebrafish strain relaxed the lack of β1a results in reduced DHPRα1S membrane expression, in impediment of tetrad formation, and also in the elimination of α1S charge movement (Schredelseker et al., 2005, PNAS). Recently we postulated a model describing the β1a subunit as an allosteric modifier of proper α1S conformation (Schredelseker/Dayal et al., 2009, JBC) and thus enabling full DHPR functionality in skeletal muscle excitation-contraction (EC) coupling.To investigate if distinct regions of β1a might be responsible for inducing the voltage-sensing function of the DHPR, we expressed different β isoforms and chimeras in isolated myotubes of relaxed larvae for patch clamp (charge movement) analysis. Quantitative immunocytochemical analyses showed that all four β isoforms (β1-β4) were able to fully target α1S into triads. Interestingly, despite full triad targeting, β3 was unable to restore considerable charge movement (Qmax, 2.53 ± 0.50 nC/μF) in contrast to the other β isoforms (Qmax, 8.86 ± 0.93 to 9.94 ± 2.06 nC/μF) upon expression in relaxed myotubes. Systematic exchanges of variable regions and conserved domains of β1a with corresponding β3 sequences revealed significantly reduced Qmax restoration with SH3 and C-terminal chimeras (Qmax, 4.02 ± 0.28 and 5.57 ± 0.74 nC/μF, respectively). In contrast, β1a/β3 chimeras with the N-terminus, HOOK and GK domain exchanged showed complete restoration of charge movement.Together, our data suggest an essential role of the conserved SH3 domain and the variable C-terminus of β1a in the induction of the voltage-sensing function of the DHPRα1S in skeletal muscle EC coupling.Grants: FWF-DK-W1101-B12, FWF-P23299-B09