Functional analysis of a frame-shift mutant of the dihydropyridine receptor pore subunit (α(1S)) expressing two complementary protein fragments

BACKGROUND: The L-type Ca(2+) channel formed by the dihydropyridine receptor (DHPR) of skeletal muscle senses the membrane voltage and opens the ryanodine receptor (RyR1). This channel-to-channel coupling is essential for Ca(2+) signaling but poorly understood. We characterized a single-base frame-shift mutant of α(1S), the pore subunit of the DHPR, that has the unusual ability to function voltage sensor for excitation-contraction (EC) coupling by virtue of expressing two complementary hemi-Ca(2+) channel fragments. RESULTS: Functional analysis of cDNA transfected dysgenic myotubes lacking α(1S) were carried out using voltage-clamp, confocal Ca(2+) indicator fluoresence, epitope immunofluorescence and immunoblots of expressed proteins. The frame-shift mutant (fs-α(1S)) expressed the N-terminal half of α(1S) (M1 to L670) and the C-terminal half starting at M701 separately. The C-terminal fragment was generated by an unexpected restart of translation of the fs-α(1S) message at M701 and was eliminated by a M701I mutation. Protein-protein complementation between the two fragments produced recovery of skeletal-type EC coupling but not L-type Ca(2+) current. DISCUSSION: A premature stop codon in the II-III loop may not necessarily cause a loss of DHPR function due to a restart of translation within the II-III loop, presumably by a mechanism involving leaky ribosomal scanning. In these cases, function is recovered by expression of complementary protein fragments from the same cDNA. DHPR-RyR1 interactions can be achieved via protein-protein complementation between hemi-Ca(2+) channel proteins, hence an intact II-III loop is not essential for coupling the DHPR voltage sensor to the opening of RyR1 channel.