Sarcoplasmic Reticulum K+ (TRIC) Channel Does Not Carry Essential Countercurrent during Ca2+ Release

AbstractThe charge translocation associated with sarcoplasmic reticulum (SR) Ca2+ 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 Ca2+ equilibrium potential and SR Ca2+ 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 Ca2+ 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 Ca2+ reloading, and caffeine-evoked Ca2+ 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 Ca2+ 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

Luminal Ca2+ Regulation of Single Cardiac Ryanodine Receptors: Insights Provided by Calsequestrin and its Mutants

The luminal Ca2+ regulation of cardiac ryanodine receptor (RyR2) was explored at the single channel level. The luminal Ca2+ and Mg2+ sensitivity of single CSQ2-stripped and CSQ2-associated RyR2 channels was defined. Action of wild-type CSQ2 and of two mutant CSQ2s (R33Q and L167H) was also compared. Two luminal Ca2+ regulatory mechanism(s) were identified. One is a RyR2-resident mechanism that is CSQ2 independent and does not distinguish between luminal Ca2+ and Mg2+. This mechanism modulates the maximal efficacy of cytosolic Ca2+ activation. The second luminal Ca2+ regulatory mechanism is CSQ2 dependent and distinguishes between luminal Ca2+ and Mg2+. It does not depend on CSQ2 oligomerization or CSQ2 monomer Ca2+ binding affinity. The key Ca2+-sensitive step in this mechanism may be the Ca2+-dependent CSQ2 interaction with triadin. The CSQ2-dependent mechanism alters the cytosolic Ca2+ sensitivity of the channel. The R33Q CSQ2 mutant can participate in luminal RyR2 Ca2+ regulation but less effectively than wild-type (WT) CSQ2. CSQ2-L167H does not participate in luminal RyR2 Ca2+ regulation. The disparate actions of these two catecholaminergic polymorphic ventricular tachycardia (CPVT)–linked mutants implies that either alteration or elimination of CSQ2-dependent luminal RyR2 regulation can generate the CPVT phenotype. We propose that the RyR2-resident, CSQ2-independent luminal Ca2+ mechanism may assure that all channels respond robustly to large (>5 μM) local cytosolic Ca2+ stimuli, whereas the CSQ2-dependent mechanism may help close RyR2 channels after luminal Ca2+ falls below ∼0.5 mM

Flux regulation of cardiac ryanodine receptor channels

The cardiac type 2 ryanodine receptor (RYR2) is activated by Ca2+-induced Ca2+ release (CICR). The inherent positive feedback of CICR is well controlled in cells, but the nature of this control is debated. Here, we explore how the Ca2+ flux (lumen-to-cytosol) carried by an open RYR2 channel influences its own cytosolic Ca2+ regulatory sites as well as those on a neighboring channel. Both flux-dependent activation and inhibition of single channels were detected when there were super-physiological Ca2+ fluxes (>3 pA). Single-channel results indicate a pore inhibition site distance of 1.2 ± 0.16 nm and that the activation site on an open channel is shielded/protected from its own flux. Our results indicate that the Ca2+ flux mediated by an open RYR2 channel in cells (∼0.5 pA) is too small to substantially regulate (activate or inhibit) the channel carrying it, even though it is sufficient to activate a neighboring RYR2 channel

Single-Channel Function of Recombinant Type 2 Inositol 1,4,5-Trisphosphate Receptor

AbstractA full-length rat type 2 inositol 1,4,5-trisphosphate (InsP3) receptor cDNA construct was generated and expressed in COS-1 cells. Targeting of the full-length recombinant type 2 receptor protein to the endoplasmic reticulum was confirmed by immunocytochemistry using isoform specific affinity-purified antibodies and InsP3R-green fluorescent protein chimeras. The receptor protein was solubilized and incorporated into proteoliposomes for functional characterization. Single-channel recordings from proteoliposomes fused into planar lipid bilayers revealed that the recombinant protein formed InsP3- and Ca2+-sensitive ion channels. The unitary conductance (∼250 pS; 220/20mM Cs+ as charge carrier), gating, InsP3, and Ca2+ sensitivities were similar to those previously described for the native type 2 InsP3R channel. However, the maximum open probability of the recombinant channel was slightly lower than that of its native counterpart. These data show that our full-length rat type 2 InsP3R cDNA construct encodes a protein that forms an ion channel with functional attributes like those of the native type 2 InsP3R channel. The possibility of measuring the function of single recombinant type 2 InsP3R is a significant step toward the use of molecular tools to define the determinants of isoform-specific InsP3R function and regulation

The Arrhythmogenic Calmodulin p.Phe142Leu Mutation Impairs C-domain Ca2+-binding but not Calmodulin-dependent Inhibition of the Cardiac Ryanodine Receptor

A number of point mutations in the intracellular Ca(2+)-sensing protein calmodulin (CaM) are arrhythmogenic, yet their underlying mechanisms are not clear. These mutations generally decrease Ca(2+) binding to CaM and impair inhibition of CaM-regulated Ca(2+) channels like the cardiac Ca(2+) release channel (ryanodine receptor, RyR2), and it appears that attenuated CaM Ca(2+) binding correlates with impaired CaM-dependent RyR2 inhibition. Here, we investigated the RyR2 inhibitory action of the CaM p.Phe142Leu mutation (F142L; numbered including the start-Met), which markedly reduces CaM Ca(2+) binding. Surprisingly, CaM-F142L had little to no aberrant effect on RyR2-mediated store overload-induced Ca(2+) release in HEK293 cells compared with CaM-WT. Furthermore, CaM-F142L enhanced CaM-dependent RyR2 inhibition at the single channel level compared with CaM-WT. This is in stark contrast to the actions of arrhythmogenic CaM mutations N54I, D96V, N98S, and D130G, which all diminish CaM-dependent RyR2 inhibition. Thermodynamic analysis showed that apoCaM-F142L converts an endothermal interaction between CaM and the CaM-binding domain (CaMBD) of RyR2 into an exothermal one. Moreover, NMR spectra revealed that the CaM-F142L-CaMBD interaction is structurally different from that of CaM-WT at low Ca(2+). These data indicate a distinct interaction between CaM-F142L and the RyR2 CaMBD, which may explain the stronger CaM-dependent RyR2 inhibition by CaM-F142L, despite its reduced Ca(2+) binding. Collectively, these results add to our understanding of CaM-dependent regulation of RyR2 as well as the mechanistic effects of arrhythmogenic CaM mutations. The unique properties of the CaM-F142L mutation may provide novel clues on how to suppress excessive RyR2 Ca(2+) release by manipulating the CaM-RyR2 interaction

Mechanism of Calsequestrin Regulation of Single Cardiac Ryanodine Receptor in normal and pathological conditions

Release of Ca(2+) from the sarcoplasmic reticulum (SR) drives contractile function of cardiac myocytes. Luminal Ca(2+) regulation of SR Ca(2+) release is fundamental not only in physiology but also in physiopathology because abnormal luminal Ca(2+) regulation is known to lead to arrhythmias, catecholaminergic polymorphic ventricular tachycardia (CPVT), and/or sudden cardiac arrest, as inferred from animal model studies. Luminal Ca(2+) regulates ryanodine receptor (RyR)2-mediated SR Ca(2+) release through mechanisms localized inside the SR; one of these involves luminal Ca(2+) interacting with calsequestrin (CASQ), triadin, and/or junctin to regulate RyR2 function. CASQ2-RyR2 regulation was examined at the single RyR2 channel level. Single RyR2s were incorporated into planar lipid bilayers by the fusion of native SR vesicles isolated from either wild-type (WT), CASQ2 knockout (KO), or R33Q-CASQ2 knock-in (KI) mice. KO and KI mice have CPVT-like phenotypes. We show that CASQ2(WT) action on RyR2 function (either activation or inhibition) was strongly influenced by the presence of cytosolic MgATP. Function of the reconstituted CASQ2(WT)–RyR2 complex was unaffected by changes in luminal free [Ca(2+)] (from 0.1 to 1 mM). The inhibition exerted by CASQ2(WT) association with the RyR2 determined a reduction in cytosolic Ca(2+) activation sensitivity. RyR2s from KO mice were significantly more sensitive to cytosolic Ca(2+) activation and had significantly longer mean open times than RyR2s from WT mice. Sensitivity of RyR2s from KI mice was in between that of RyR2 channels from KO and WT mice. Enhanced cytosolic RyR2 Ca(2+) sensitivity and longer RyR2 open times likely explain the CPVT-like phenotype of both KO and KI mice