In the heart, reliable activation of Ca2+ release from the sarcoplasmic reticulum during the plateau of the ventricular action potential requires synchronous opening of multiple CaV1.2 channels. Yet the mechanisms that coordinate this simultaneous opening during every heartbeat are unclear. Here, we demonstrate that CaV1.2 channels form clusters that undergo dynamic, reciprocal, allosteric interactions. This ‘functional coupling’ facilitates Ca2+ influx by increasing activation of adjoined channels and occurs through C-terminal-to-C-terminal interactions. These interactions are initiated by binding of incoming Ca2+ to calmodulin (CaM) and proceed through Ca2+/CaM binding to the CaV1.2 pre-IQ domain. Coupling fades as [Ca2+]i decreases, but persists longer than the current that evoked it, providing evidence for ‘molecular memory’. Our findings suggest a model for CaV1.2 channel gating and Ca2+-influx amplification that unifies diverse observations about Ca2+ signaling in the heart, and challenges the long-held view that voltage-gated channels open and close independently.DOI: http://dx.doi.org/10.7554/eLife.05608.001
Rationale L-type Ca2+ (CaV1.2) channels shape the cardiac action potential waveform and are essential for excitation-contraction coupling in heart. A gain-of-function G406R mutation in a cytoplasmic loop of CaV1.2 channels causes long QT syndrome 8 (LQT8), a disease also known as Timothy syndrome. However, the mechanisms by which this mutation enhances CaV1.2-LQT8 currents and generates lethal arrhythmias are unclear. Objective To test the hypothesis that the anchoring protein AKAP150 modulates CaV1.2-LQT8 channel gating in ventricular myocytes. Methods and Results Using a combination of molecular, imaging, and electrophysiological approaches, we discovered that CaV1.2-LQT8 channels are abnormally coupled to AKAP150. A pathophysiological consequence of forming this aberrant ion channel-anchoring protein complex is enhanced CaV1.2-LQT8 currents. This occurs through a mechanism whereby the anchoring protein functions like a subunit of CaV1.2-LQT8 channels that stabilizes the open conformation and augments the probability of coordinated openings of these channels. Ablation of AKAP150 restores normal gating in CaV1.2-LQT8 channels and protects the heart from arrhythmias. Conclusion We propose that AKAP150-dependent changes in CaV1.2-LQT8 channel gating may constitute a novel general mechanism for CaV1.2-driven arrhythmias.
A-kinase anchoring proteins (AKAPs) tether the cAMP-dependent protein kinase (PKA) to intracellular sites where they preferentially phosphorylate target substrates. Most AKAPs exhibit nanomolar affinity for the regulatory (RII) subunit of the type II PKA holoenzyme, whereas dual-specificity anchoring proteins also bind the type I (RI) regulatory subunit of PKA with 10-100-fold lower affinity. A range of cellular, biochemical, biophysical, and genetic approaches comprehensively establish that sphingosine kinase interacting protein (SKIP) is a truly type I-specific AKAP. Mapping studies located anchoring sites between residues 925-949 and 1,140-1,175 of SKIP that bind RI with dissociation constants of 73 and 774 nM, respectively. Molecular modeling and site-directed mutagenesis approaches identify Phe 929 and Tyr 1,151 as RI-selective binding determinants in each anchoring site. SKIP complexes exist in different states of RI-occupancy as single-molecule pulldown photobleaching experiments show that 41 AE 10% of SKIP sequesters two YFP-RI dimers, whereas 59 AE 10% of the anchoring protein binds a single YFP-RI dimer. Imaging, proteomic analysis, and subcellular fractionation experiments reveal that SKIP is enriched at the inner mitochondrial membrane where it associates with a prominent PKA substrate, the coiled-coil helix protein ChChd3.
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