Functional analysis of a frame-shift mutant of the dihydropyridine receptor pore subunit (α1S) expressing two complementary protein fragments

Chris A Ahern; Paola Vallejo; Lindsay Mortenson; Roberto Coronado

doi:10.1186/1472-6793-1-15

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Functional analysis of a frame-shift mutant of the dihydropyridine receptor pore subunit (α1S) expressing two complementary protein fragments

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Functional analysis of a frame-shift mutant of the dihydropyridine receptor pore subunit (α1S) expressing two complementary protein fragments

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

BMC physiology, Vol.1(1), pp.15-15

2001

DOI: 10.1186/1472-6793-1-15

PMCID: PMC64647

PMID: 11806762

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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 alpha1S, 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 alpha1S were carried out using voltage-clamp, confocal Ca2+ indicator fluoresence, epitope immunofluorescence and immunoblots of expressed proteins. The frame-shift mutant (fs-alpha1S) expressed the N-terminal half of alpha1S (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-alpha1S 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.

Details

Title: Subtitle: Functional analysis of a frame-shift mutant of the dihydropyridine receptor pore subunit (α1S) expressing two complementary protein fragments
Creators: Chris A Ahern - University of Iowa, Molecular Physiology and Biophysics
Paola Vallejo - Department of Physiology, University of Wisconsin School of Medicine, Madison, WI 53706, USA
Lindsay Mortenson - Department of Physiology, University of Wisconsin School of Medicine, Madison, WI 53706, USA
Roberto Coronado - Department of Physiology, University of Wisconsin School of Medicine, Madison, WI 53706, USA
Resource Type: Journal article
Publication Details: BMC physiology, Vol.1(1), pp.15-15
Publisher: BioMed Central
DOI: 10.1186/1472-6793-1-15
PMID: 11806762
PMCID: PMC64647
ISSN: 1472-6793
eISSN: 1472-6793
Language: English
Date published: 2001
Academic Unit: Molecular Physiology and Biophysics; Iowa Neuroscience Institute
Record Identifier: 9984071719602771

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