Research article

Functional analysis of a frame-shift mutant of the dihydropyridine receptor pore subunit (α_1S) expressing two complementary protein fragments

Chris A Ahern , Paola Vallejo , Lindsay Mortenson and Roberto Coronado

Department of Physiology, University of Wisconsin School of Medicine, Madison, WI 53706, USA

author email corresponding author email

BMC Physiology 2001, 1:15doi:10.1186/1472-6793-1-15

Published:	31 December 2001

Abstract

Background

The L-type Ca²⁺ 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 Ca²⁺ 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-Ca²⁺ channel fragments.

Results

Functional analysis of cDNA transfected dysgenic myotubes lacking α_1S were carried out using voltage-clamp, confocal Ca²⁺ 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 Ca²⁺ 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-Ca²⁺ channel proteins, hence an intact II-III loop is not essential for coupling the DHPR voltage sensor to the opening of RyR1 channel.