Regulation of L-type calcium channel by phospholemman in cardiac myocytes.
We evaluated whether phospholemman (PLM) regulates L-type Ca(2+) current (ICa) in mouse ventricular myocytes. Expression of α1-subunit of L-type Ca(2+) channels between wild-type (WT) and PLM knockout (KO) hearts was similar. Compared to WT myocytes, peak ICa (at -10 mV) from KO myocytes was ~41% larger, the inactivation time constant (τ(inact)) of ICa was ~39% longer, but deactivation time constant (τ(deact)) was similar. In the presence of isoproterenol (1 μM), peak ICa was ~48% larger and τ(inact) was ~144% higher in KO myocytes. With Ba(2+) as the permeant ion, PLM enhanced voltage-dependent inactivation but had no effect on τ(deact). To dissect the molecular determinants by which PLM regulated ICa, we expressed PLM mutants by adenovirus-mediated gene transfer in cultured KO myocytes. After 24h in culture, KO myocytes expressing green fluorescent protein (GFP) had significantly larger peak ICa and longer τ(inact) than KO myocytes expressing WT PLM; thereby independently confirming the observations in freshly isolated myocytes. Compared to KO myocytes expressing GFP, KO myocytes expressing the cytoplasmic domain truncation mutant (TM43), the non-phosphorylatable S68A mutant, the phosphomimetic S68E mutant, and the signature PFXYD to alanine (ALL5) mutant all resulted in lower peak ICa. Expressing PLM mutants did not alter expression of α1-subunit of L-type Ca(2+) channels in cultured KO myocytes. Our results suggested that both the extracellular PFXYD motif and the transmembrane domain of PLM but not the cytoplasmic tail were necessary for regulation of peak ICa amplitude. We conclude that PLM limits Ca(2+) influx in cardiac myocytes by reducing maximal ICa and accelerating voltage-dependent inactivation.
Center of Translational Medicine, Temple University School of Medicine, Philadelphia, PA, USA.
Xue-Qian
XQ
Zhang
Center of Translational Medicine, Temple University School of Medicine, Philadelphia, PA, USA.
JuFang
J
Wang
Center of Translational Medicine, Temple University School of Medicine, Philadelphia, PA, USA.
Jianliang
J
Song
Center of Translational Medicine, Temple University School of Medicine, Philadelphia, PA, USA.
Joseph
J
Rabinowitz
Cardiovascular Research Center, Temple University School of Medicine, Philadelphia, PA, USA.
Xiongwen
X
Chen
Cardiovascular Research Center, Temple University School of Medicine, Philadelphia, PA, USA.
Steven R
SR
Houser
Department of Cellular and Molecular Physiology, The Pennsylvania State University College of Medicine, Hershey, PA, USA.
Blaise Z
BZ
Peterson
Cardiovascular Division, Department of Internal Medicine, University of Virginia Health Sciences Center, Charlottesville, VA, USA.
Amy L
AL
Tucker
Cardiovascular Research Center, Temple University School of Medicine, Philadelphia, PA, USA.
Arthur M
AM
Feldman
Center of Translational Medicine, Temple University School of Medicine, Philadelphia, PA, USA. Electronic address: joseph.cheung@tuhs.temple.edu.
Joseph Y
JY
Cheung
D000256:Adenoviridae / Q000378:metabolism; D020816:Amino Acid Motifs; D000818:Animals; D020746:Calcium Channels, L-Type / Q000378:metabolism*; D002478:Cells, Cultured; D003593:Cytoplasm / Q000737:chemistry; D004285:Dogs; D006352:Heart Ventricles / Q000187:drug effects / Q000378:metabolism; D015640:Ion Channel Gating / Q000187:drug effects; D007545:Isoproterenol / Q000494:pharmacology; D008565:Membrane Proteins / Q000737:chemistry / Q000378:metabolism*; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D050505:Mutant Proteins / Q000378:metabolism; D032383:Myocytes, Cardiac / Q000187:drug effects / Q000378:metabolism*; D010750:Phosphoproteins / Q000737:chemistry / Q000378:metabolism*; D010768:Phosphoserine / Q000378:metabolism; D017434:Protein Structure, Tertiary; D013329:Structure-Activity Relationship
C514350:CACNA1C protein, mouse; D020746:Calcium Channels, L-Type; D008565:Membrane Proteins; D050505:Mutant Proteins; D010750:Phosphoproteins; C069141:phospholemman; D010768:Phosphoserine; D007545:Isoproterenol
Arrhythmia; Ca(2+) channels; FXYD1; Phospholemman