title: Functional analysis of a frame-shift mutant of the dihydropyridine receptor pore subunit (alpha1S) expressing two complementary protein fragments.
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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Department of Physiology, University of Wisconsin School of Medicine, Madison, WI 53706, USA. cahern@physiology.wisc.edu
authors.forename: C A
authors.initials: CA
authors.lastname: Ahern
authors.forename: P
authors.initials: P
authors.lastname: Vallejo
authors.forename: L
authors.initials: L
authors.lastname: Mortenson
authors.forename: R
authors.initials: R
authors.lastname: Coronado
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002118:Calcium / Q000378:metabolism; D020746:Calcium Channels, L-Type / Q000737:chemistry / Q000235:genetics* / Q000502:physiology*; D002478:Cells, Cultured; D016368:Frameshift Mutation; D051379:Mice; D008969:Molecular Sequence Data; D009119:Muscle Contraction; D018485:Muscle Fibers, Skeletal / Q000502:physiology; D018482:Muscle, Skeletal / Q000378:metabolism / Q000502:physiology*; D018408:Patch-Clamp Techniques; D021122:Protein Subunits
chemical_list: D020746:Calcium Channels, L-Type; D021122:Protein Subunits; D002118:Calcium