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1.
J Gen Physiol ; 118(5): 495-508, 2001 Nov.
Article in English | MEDLINE | ID: mdl-11696608

ABSTRACT

A defining property of L-type Ca(2+) channels is their potentiation by both 1,4-dihydropyridine agonists and strong depolarization. In contrast, non-L-type channels are potentiated by neither agonist nor depolarization, suggesting that these two processes may by linked. In this study, we have tested whether the mechanisms of agonist- and depolarization-induced potentiation in the cardiac L-type channel (alpha(1C)) are linked. We found that the mutant L-type channel GFP-alpha(1C)(TQ-->YM), bearing the mutations T1066Y and Q1070M, was able to undergo depolarization-induced potentiation but not potentiation by agonist. Conversely, the chimeric channel GFP-CACC was potentiated by agonist but not by strong depolarization. These data indicate that the mechanisms of agonist- and depolarization-induced potentiation of alpha(1C) are distinct. Since neither GFP-CACC nor GFP-CCAA was potentiated significantly by depolarization, no single repeat of alpha(1C) appears to be responsible for depolarization-induced potentiation. Surprisingly, GFP-CACC displayed a low estimated open probability similar to that of the alpha(1C), but could not support depolarization-induced potentiation, demonstrating that a relatively low open probability alone is not sufficient for depolarization-induced potentiation to occur. Thus, depolarization-induced potentiation may be a global channel property requiring participation from all four homologous repeats.


Subject(s)
Calcium Channel Blockers/pharmacology , Calcium Channels, L-Type/physiology , Dihydropyridines/pharmacology , Heart/physiology , Action Potentials , Animals , Electrophysiology , Membrane Potentials , Mice , Mutagenesis, Site-Directed , Point Mutation , Polymerase Chain Reaction
2.
Proc Natl Acad Sci U S A ; 98(10): 5892-7, 2001 May 08.
Article in English | MEDLINE | ID: mdl-11320225

ABSTRACT

The II-III loop of the skeletal muscle dihydropyridine receptor (DHPR) alpha(1S) subunit is responsible for bidirectional-signaling interactions with the ryanodine receptor (RyR1): transmitting an orthograde, excitation-contraction (EC) coupling signal to RyR1 and receiving a retrograde, current-enhancing signal from RyR1. Previously, several reports argued for the importance of two distinct regions of the skeletal II-III loop (residues R681-L690 and residues L720-Q765, respectively), claiming for each a key function in DHPR-RyR1 communication. To address whether residues 720-765 of the II-III loop are sufficient to enable skeletal-type (Ca(2+) entry-independent) EC coupling and retrograde interaction with RyR1, we constructed a green fluorescent protein (GFP)-tagged chimera (GFP-SkLM) having rabbit skeletal (Sk) DHPR sequence except for a II-III loop (L) from the DHPR of the house fly, Musca domestica (M). The Musca II-III loop (75% dissimilarity to alpha(1S)) has no similarity to alpha(1S) in the regions R681-L690 and L720-Q765. GFP-SkLM expressed in dysgenic myotubes (which lack endogenous alpha(1S) subunits) was unable to restore EC coupling and displayed strongly reduced Ca(2+) current densities despite normal surface expression levels and correct triad targeting (colocalization with RyR1). Introducing rabbit alpha(1S) residues L720-L764 into the Musca II-III loop of GFP-SkLM (substitution for Musca DHPR residues E724-T755) completely restored bidirectional coupling, indicating its dependence on alpha(1S) loop residues 720-764 but its independence from other regions of the loop. Thus, 45 alpha(1S)-residues embedded in a very dissimilar background are sufficient to restore bidirectional coupling, indicating that these residues may be a site of a protein-protein interaction required for bidirectional coupling.


Subject(s)
Calcium Channels, L-Type/physiology , Muscle, Skeletal/physiology , Amino Acid Sequence , Animals , Calcium/metabolism , Calcium Channels, L-Type/chemistry , Calcium Channels, L-Type/genetics , Cells, Cultured , DNA, Complementary , Molecular Sequence Data , Muscle, Skeletal/chemistry , Muscle, Skeletal/metabolism , Patch-Clamp Techniques , Rabbits , Recombinant Fusion Proteins/genetics , Sequence Homology, Amino Acid
3.
J Biol Chem ; 275(39): 29935-7, 2000 Sep 29.
Article in English | MEDLINE | ID: mdl-10915779

ABSTRACT

A peptide corresponding to residues 681-690 of the II-III loop of the skeletal muscle dihydropyridine receptor alpha(1) subunit (DHPR, alpha(1S)) has been reported to activate the skeletal muscle ryanodine receptor (RyR1) in vitro. Within this region of alpha(1S), a cluster of basic residues, Arg(681)-Lys(685), was previously reported to be indispensable for the activation of RyR1 in microsomal preparations and lipid bilayers. We have used an intact alpha(1S) subunit with scrambled sequence in this region of the II-III loop (alpha(1S)-scr) to test the importance of residues 681-690 and the basic motif for skeletal-type excitation-contraction (EC) coupling and retrograde signaling in vivo. When expressed in dysgenic myotubes (which lack endogenous alpha(1S)), alpha(1S)-scr restored calcium currents that were indistinguishable, in current density and voltage dependence, from those restored by wild-type alpha(1S). The scrambled DHPR also rescued skeletal-type EC coupling, as indicated by electrically evoked contractions in the presence of 0.5 mm Cd(2+) and 0.1 mm La(3+). Furthermore, the release of intracellular Ca(2+), as assayed by the indicator dye, Fluo-3, had similar kinetics and voltage dependence for alpha(1S) and alpha(1S)-scr. These data suggest that residues 681-690 of the alpha(1S) II-III loop are not essential in muscle cells for normal functioning of the DHPR, including skeletal-type EC coupling and retrograde signaling.


Subject(s)
Muscle Contraction/physiology , Muscle, Skeletal/physiology , Ryanodine Receptor Calcium Release Channel/metabolism , Amino Acid Sequence , Animals , Calcium/metabolism , Mice , Models, Molecular , Oligopeptides/chemistry , Oligopeptides/genetics , Oligopeptides/metabolism , Peptide Fragments/chemistry , Peptide Fragments/genetics , Peptide Fragments/metabolism , Protein Structure, Secondary , Ryanodine Receptor Calcium Release Channel/chemistry , Ryanodine Receptor Calcium Release Channel/genetics , Signal Transduction
4.
Vet Rec ; 126(10): 248-9, 1990 Mar 10.
Article in English | MEDLINE | ID: mdl-2321341
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