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Ca v 1.2 channel oligomerization amplifies Ca 2+ influx during β‐adrenergic stimulation
Author(s) -
Dixon Rose Ellen,
Santana Luis Fernando
Publication year - 2016
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.30.1_supplement.1274.9
Subject(s) - chemistry , biophysics , forskolin , stimulation , agonist , adenylyl cyclase , bimolecular fluorescence complementation , allosteric regulation , electrophysiology , signal transduction , neuroscience , biochemistry , biology , receptor , gene
L‐type Ca v 1.2 channels are ubiquitously expressed in the surface membranes of excitable cells where they regulate myriad processes including muscle contraction, neuronal excitability, hormone secretion and gene expression. Recently, we reported that Ca v 1.2 channels form clusters that undergo dynamic, reciprocal, allosteric interactions. This ‘functional coupling’ facilitates Ca 2+ influx by increasing activity ( NP o ) of adjoined channels and occurs through C‐terminal‐to‐C‐terminal interactions. Channel‐to‐channel interactions are initiated by binding of incoming Ca 2+ to CaM and proceed through Ca 2+ /CaM binding to the Ca v 1.2 pre‐IQ domain. FRET experiments revealed that coupling fades as [Ca 2+ ] i decreases, but persists longer than the current that evoked it, providing evidence for ‘molecular memory’. This creates a ‘primed state’ which may have a role during repetitive stimulations, such as those that occur during periods of increased heart rate – stimulated for instance by exercise or the fight‐or‐flight response. This ‘memory’ could be beneficial in generating a positive inotropic effect. In the present study we used FRET, Bi molecular F luorescence C omplementation (BiFC), electrophysiology and Ca 2+ imaging to investigate the effects of β‐adrenergic signaling on Ca v 1.2 interactions. Optical and electrophysiological examination of single channel activity in mouse ventricular myocytes or transiently transfected tsA‐201 cells revealed increased coupling of Ca v 1.2 channels after application of the β‐agonist isoproterenol (iso; 100 nM) or the activator of adenylyl cyclase, forskolin (10 μM). BiFC experiments in tsA‐201 cells co‐transfected with Ca v 1.2 channels tagged with the N‐ or C‐terminal half of the split Venus fluorescent protein (Ca v 1.2‐VN and Ca v 1.2 ‐VC) revealed increased channel‐channel interactions stimulated by iso or forskolin and inhibited by the PKA inhibitor (PKI). These data suggest that β‐adrenergic stimulation amplifies Ca v 1.2 channel activity by increasing/prolonging Ca v 1.2 channel interactions. Support or Funding Information This work was supported by grants from the AHA (15SDG25560035) and NIH (HL085870 and HL085686).