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Sarcoplasmic reticulum K(+) (TRIC) channel does not carry essential countercurrent during Ca(2+) release.

ABSTRACT: The charge translocation associated with sarcoplasmic reticulum (SR) Ca(2+) efflux is compensated for by a simultaneous SR K(+) influx. This influx is essential because, with no countercurrent, the SR membrane potential (Vm) would quickly (<1 ms) reach the Ca(2+) equilibrium potential and SR Ca(2+) release would cease. The SR K(+) trimeric intracellular cation (TRIC) channel has been proposed to carry the essential countercurrent. However, the ryanodine receptor (RyR) itself also carries a substantial K(+) countercurrent during release. To better define the physiological role of the SR K(+) channel, we compared SR Ca(2+) transport in saponin-permeabilized cardiomyocytes before and after limiting SR K(+) channel function. Specifically, we reduced SR K(+) channel conduction 35 and 88% by replacing cytosolic K(+) for Na(+) or Cs(+) (respectively), changes that have little effect on RyR function. Calcium sparks, SR Ca(2+) reloading, and caffeine-evoked Ca(2+) release amplitude (and rate) were unaffected by these ionic changes. Our results show that countercurrent carried by SR K(+) (TRIC) channels is not required to support SR Ca(2+) release (or uptake). Because K(+) enters the SR through RyRs during release, the SR K(+) (TRIC) channel most likely is needed to restore trans-SR K(+) balance after RyRs close, assuring SR Vm stays near 0 mV.

SUBMITTER: Guo T

PROVIDER: S-EPMC3762336 | biostudies-literature | 2013 Sep

REPOSITORIES: biostudies-literature

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Sarcoplasmic reticulum K(+) (TRIC) channel does not carry essential countercurrent during Ca(2+) release.

Guo Tao T Nani Alma A Shonts Stephen S Perryman Matthew M Chen Haiyan H Shannon Thomas T Gillespie Dirk D Fill Michael M

Biophysical journal 20130901 5

The charge translocation associated with sarcoplasmic reticulum (SR) Ca(2+) efflux is compensated for by a simultaneous SR K(+) influx. This influx is essential because, with no countercurrent, the SR membrane potential (Vm) would quickly (<1 ms) reach the Ca(2+) equilibrium potential and SR Ca(2+) release would cease. The SR K(+) trimeric intracellular cation (TRIC) channel has been proposed to carry the essential countercurrent. However, the ryanodine receptor (RyR) itself also carries a subst ...[more]

PMID: 24010658

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Project description:Experiments were performed to characterize the properties of the intrinsic Ca(2+) buffers in the sarcoplasmic reticulum (SR) of cut fibers from frog twitch muscle. The concentrations of total and free calcium ions within the SR ([Ca(T)](SR) and [Ca(2+)](SR)) were measured, respectively, with the EGTA/phenol red method and tetramethylmurexide (a low affinity Ca(2+) indicator). Results indicate SR Ca(2+) buffering was consistent with a single cooperative-binding component or a combination of a cooperative-binding component and a linear binding component accounting for 20% or less of the bound Ca(2+). Under the assumption of a single cooperative-binding component, the most likely resting values of [Ca(2+)](SR) and [Ca(T)](SR) are 0.67 and 17.1 mM, respectively, and the dissociation constant, Hill coefficient, and concentration of the Ca-binding sites are 0.78 mM, 3.0, and 44 mM, respectively. This information can be used to calculate a variable proportional to the Ca(2+) permeability of the SR, namely d[Ca(T)](SR)/dt ÷ [Ca(2+)](SR) (denoted release permeability), in experiments in which only [Ca(T)](SR) or [Ca(2+)](SR) is measured. In response to a voltage-clamp step to -20 mV at 15°C, the release permeability reaches an early peak followed by a rapid decline to a quasi-steady level that lasts ~50 ms, followed by a slower decline during which the release permeability decreases by at least threefold. During the quasi-steady level of release, the release amplitude is 3.3-fold greater than expected from voltage activation alone, a result consistent with the recruitment by Ca-induced Ca(2+) release of 2.3 SR Ca(2+) release channels neighboring each channel activated by its associated voltage sensor. Release permeability at -60 mV increases as [Ca(T)](SR) decreases from its resting physiological level to ~0.1 of this level. This result argues against a release termination mechanism proposed in mammalian muscle fibers in which a luminal sensor of [Ca(2+)](SR) inhibits release when [Ca(T)](SR) declines to a low level.

Project description:BackgroundCatecholaminergic polymorphic ventricular tachycardia (CPVT) is a potentially lethal cardiac arrhythmia syndrome triggered by catecholamines released during exercise, stress, or sudden emotion. Variants in the calsequestrin-2 gene (CASQ2), encoding the major calcium (Ca) binding protein in the sarcoplasmic reticulum (SR), are the second most common cause of CPVT. Recently, several CASQ2 gene variants, such as CASQ2-K180R, have been linked to an autosomal dominant form of Casq2-linked CPVT (CPVT2), but the underlying mechanism is not known.MethodsA K180R mouse model was generated using CRIPSR/Cas9. Heterozygous and homozygous K180R mice were studied using telemetry ECG recordings in vivo. Ventricular cardiomyocytes were isolated and studied using fluorescent Ca indicators and patch clamp. Expression levels and localization of SR Ca-handling proteins were evaluated using Western blotting and immunostaining. Intra-SR Ca kinetics were quantified using low-affinity Ca indicators.ResultsK180R mice exhibit an autosomal dominant CPVT phenotype following exercise or catecholamine stress. Upon catecholamine stress, K180R ventricular cardiomyocytes exhibit increased spontaneous SR Ca release events, triggering delayed afterdepolarizations and spontaneous beats. K180R had no effect on levels of Casq2, Casq2 polymers, or other SR Ca-handling proteins. Intra-SR Ca measurements revealed that K180R impaired dynamic intra-SR Ca buffering, resulting in a more rapid rise of free Ca in the SR during diastole. Steady-state SR Ca buffering and total SR Ca content were not changed. Consistent with the reduced dynamic intra-SR buffering, K180R causes reduced SR Ca release refractoriness.ConclusionsCASQ2-K180R causes CPVT2 via a heretofore unknown mechanism that differs from CASQ2 variants associated with autosomal recessive CPVT2. Unlike autosomal recessive CASQ2 variants, K180R impairs the dynamic buffering of Ca within the SR without affecting total SR Ca content or Casq2 protein levels. Our data provide insight into the molecular mechanism underlying autosomal dominant CPVT2.

Dataset Information

Sarcoplasmic reticulum K(+) (TRIC) channel does not carry essential countercurrent during Ca(2+) release.

Publications

Sarcoplasmic reticulum K(+) (TRIC) channel does not carry essential countercurrent during Ca(2+) release.

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