Stable incorporation versus dynamic exchange of β subunits in a native Ca2+ channel complex

Voltage-gated Ca2+ channels are multi-subunit membrane proteins that transduce depolarization into cellular functions such as excitation-contraction coupling in muscle or neurotransmitter release in neurons. The auxiliary b subunits function in membrane targeting of the channel and modulation of its gating properties. However, whether b subunits can reversibly interactwith, and thus differentially modulate, channels in the membrane is still unresolved. In the present study we applied fluorescence recovery after photobleaching (FRAP) of GFP-tagged a1 and b subunits expressed in dysgenic myotubes to study the relative dynamics of these Ca2+ channel subunits for the first time in a native functional signaling complex. Identical fluorescence recovery rates of both subunits indicate stable interactions, distinct recovery rates indicate dynamic interactions. Whereas the skeletal muscle β1a isoform formed stable complexes with CaV1.1 and CaV1.2, the non-skeletal muscle β2a and β4b isoforms dynamically interacted with both a1 subunits. Neither replacing the I-II loop of CaV1.1 with that of CaV2.1, nor deletions inthe proximal I-II loop, known to change the orientation of b relative to the a1 subunit, altered the specific dynamic properties of the b subunits. In contrast, a single residue substitution in the a interaction pocket of β1aM293A increased the FRAP rate threefold. Taken together,these findings indicate that in skeletal muscle triads the homologous β1a subunit forms a stable complex, whereas the heterologous β2a and β4b subunits form dynamic complexes with the Ca2+ channel. The distinct binding properties are not determined by differences in the I-II loop sequences of the a1 subunits, but are intrinsic properties of the b subunit isoforms. © 2013. Published by The Company of Biologists Ltd.