Increased Coupled Gating of L-Type Ca ²⁺ Channels During Hypertension and Timothy Syndrome

Abstract

Key Words: Cav1.2 channels Ⅲ PKC Ⅲ calmodulin Ⅲ arrhythmias V oltage-gated L-Type (Cav1.2) Ca 2ϩ channels are expressed in the surface membrane of neurons and muscle cells, where they regulate multiple processes including excitability, contraction, gene expression, and memory storage. [1][2][3][4] Recent studies have revealed an unexpected feature of Cav1.2 channels: their activity (ie, open probability, P o ) varies within the cell membrane. [5][6][7] Low activity Cav1.2 channels open randomly and infrequently at rest, producing brief elevations in intracellular Ca 2ϩ concentration ([Ca 2ϩ ] i ) called "Ca 2ϩ sparklets." 8 In contrast, small clusters of Cav1.2 channels can function in a high open probability mode that generates localized zones of relatively high Ca 2ϩ influx ("persistent Ca 2ϩ sparklets"). Although targeting of protein kinase (PK)C␣ to the surface membrane by the kinase anchoring protein AKAP150 (the rodent ortholog of human AKAP79) increases the probability of persistent Cav1.2 channel activity, 9 the mechanisms by which small clusters of these channels open concertedly are unknown. Here, we used fluorescence resonance energy transfer (FRET) approaches in combination with optical and electrophysiological recordings of Ca 2ϩ influx via Cav1.2 channels to investigate this important issue. Our data indicate that activators of PKC␣, calmodulin (CaM) antagonists, or a specific Cav1.2 channel mutation that causes arrhythmias and autism in humans with the Timothy syndrome (TS) move the ubiquitous Ca 2ϩ -binding protein CaM from the IQ domain in the C-terminal tail of the channel. This induces functional interactions between nearby Cav1.2 channel via their C termini that could lead to coupled gating of these channels. MethodsAn expanded Methods section is available in the Online Data Supplement at http://circres.ahajournals.org. Isolation of Arterial and Adult and Neonatal Cardiac MyocytesMice and rats were euthanized as approved by the University of Washington Institutional Animal Care and Use Committee. Adult arterial myocytes and neonatal cardiac myocytes were prepared as described previously. 5,10 Cav1.2 and Calmodulin Constructs and Inducible PKC␣ Translocation System and Their Expression in tsA-201 CellsWe transfected tsA-201 cells using JetPEI (Polyplus Transfection) with plasmids encoding calcium channel pore-forming and accessory subunits as well as CaM. We generated the rabbit homolog of the human TS Cav1.2 (G436R, rabbit; G406R human Coupled Markov Chain ModelWe implemented a coupled Markov chain model in Matlab to determine the coupling coefficient () among Cav1.2 channels underlying membrane currents and Ca 2ϩ sparklet sites. The model was originally developed by Chung and Kennedy 14 to analyze individual records of partially coupled GABA-activated chloride channels. Electrophysiology, Confocal, and Total Internal Reflection Fluorescence MicroscopyVoltage-clamp experiments were performed using standard patchclamp techniques. Total internal reflection fluorescence (TIRF) images were...

Associations of Variants in theCACNA1AandCACNA1CGenes With Longitudinal Blood Pressure Changes and Hypertension Incidence: The GenSalt Study

“…6 For example, upregulation of VDCCs expression may contribute to Ca 2+ imbalances and result in hypertension. 7,8 Tajada et al 20 found that the increased Ca 2+ influx finally led to abnormal arterial tone due to the increased VDCCs function in hypertensive mice. .…”

Local control of TRPV4 channels by AKAP150-targeted PKC in arterial smooth muscle

“…Thus, multiple Ca V 1.2 channels would have to open simultaneously to produce the same local Ca 2+ signal as that of a single TRPV4 channel in an arterial myocyte. Although clusters of 2-6 Ca V 1.2 channels can open coordinately (Navedo et al, 2010), even these relatively rare coupled gating events would fall short of the Ca 2+ flux through a single TRPV4 channel in arterial myocytes. The high flux of Ca 2+ through individual TRPV4 channels allows for tight functional coupling between these channels and RyRs over a wider range of distances than for Ca V 1.2 channels in arterial myocytes.…”

“…Sparklets were detected and analyzed using custom software written in MATLAB (Navedo et al, 2010). Fluorescence intensity values were converted to nanomolar units as described previously (Navedo et al, 2005(Navedo et al, , 2006.…”

Nitric Oxide–Dependent Feedback Loop Regulates Transient Receptor Potential Vanilloid 4 (TRPV4) Channel Cooperativity and Endothelial Function in Small Pulmonary Arteries

“…Coupling coefficients for the coupling among TRPV4 channels at a cluster were determined using a coupled Markov chain model in MATLAB, as previously described 5, 31, 32, 33, 34. [F‐F 0 ]/F 0 traces showing steady baseline (ie, at least 30 s duration) were selected for analyzing the cooperative gating of TRPV4 channels.…”