Daniel A. Pasek

Calmodulin Binding and Inhibition of Cardiac Muscle Calcium Release Channel (Ryanodine Receptor)

Metabolically 35S-labeled calmodulin (CaM) was used to determine the CaM binding properties of the cardiac ryanodine receptor (RyR2) and to identify potential channel domains for CaM binding. In addition, regulation of RyR2 by CaM was assessed in [3H]ryanodine binding and single-channel measurements. Cardiac sarcoplasmic reticulum vesicles bound approximately four CaM molecules per RyR2 tetramer in the absence of Ca2+; in the presence of 100 μmCa2+, the vesicles bound 7.5 CaM molecules per tetramer. Purified RyR2 bound approximately four [35S]CaM molecules per RyR tetramer, both in the presence and absence of Ca2+. At least four CaM binding domains were identified in [35S]CaM overlays of fusion proteins spanning the full-length RyR2. The affinity (but not the stoichiometry) of CaM binding was altered by redox state as controlled by the presence of either GSH or GSSG. Inhibition of RyR2 activity by CaM was influenced by Ca2+ concentration, redox state, and other channel modulators. Parallel experiments with the skeletal muscle isoform showed major differences in the CaM binding properties and regulation by CaM of the skeletal and cardiac ryanodine receptors.

Regulation of Skeletal Muscle Ca2+ Release Channel (Ryanodine Receptor) by Ca2+ and Monovalent Cations and Anions

The effects of ionic composition and strength on rabbit skeletal muscle Ca2+ release channel (ryanodine receptor) activity were investigated in vesicle-45Ca2+ flux, single channel and [3H]ryanodine binding measurements. In <0.01 μM Ca2+ media, the highest 45Ca2+ efflux rate was measured in 0.25 M choline-Cl medium followed by 0.25 M KCl, choline 4-morpholineethanesulfonic acid (Mes), potassium 1,4-piperazinediethanesulfonic acid (Pipes), and K-Mes medium. In all five media, the 45Ca2+ efflux rates were increased when the free [Ca2+] was raised from <0.01 μM to 20 μM and decreased as the free [Ca2+] was further increased to 1 mM. An increase in [KCl] augmented Ca2+-gated single channel activity and [3H]ryanodine binding. In [3H]ryanodine binding measurements, bell-shaped Ca2+ activation/inactivation curves were obtained in media containing different monovalent cations (Li+, Na+, K+, Cs+, and choline+) and anions (Cl−, Mes−, and Pipes−). In choline-Cl medium, substantial levels of [3H]ryanodine binding were observed at [Ca2+] <0.01 μM. Replacement of Cl− by Mes− or Pipes− reduced [3H]ryanodine binding levels at all [Ca2+]. In all media, the Ca2+-dependence of [3H]ryanodine binding could be well described assuming that the skeletal muscle ryanodine receptor possesses cooperatively interacting high-affinity Ca2+ activation and low-affinity Ca2+ inactivation sites. AMP primarily affected [3H]ryanodine binding by decreasing the apparent affinity of the Ca2+ inactivation site(s) for Ca2+, while caffeine increased the apparent affinity of the Ca2+ activation site for Ca2+. Competition studies indicated that ionic composition affected Ca2+-dependent receptor activity by at least three different mechanisms: (i) competitive binding of Mg2+ and monovalent cations to the Ca2+ activation sites, (ii) binding of divalent cations to the Ca2+ inactivation sites, and (iii) binding of anions to specific anion regulatory sites.

Molecular Basis of Calmodulin Binding to Cardiac Muscle Ca2+ Release Channel (Ryanodine Receptor)

Calmodulin (CaM) is a ubiquitous Ca2+-binding protein that regulates the ryanodine receptors (RyRs) by direct binding. CaM inhibits the skeletal muscle ryanodine receptor (RyR1) and cardiac muscle receptor (RyR2) at >1 μm Ca2+ but activates RyR1 and inhibits RyR2 at <1 μm Ca2+. Here we tested whether CaM regulates RyR2 by binding to a highly conserved site identified previously in RyR1. Deletion of RyR2 amino acid residues 3583–3603 resulted in background [35S]CaM binding levels. In single channel measurements, deletion of the putative CaM binding site eliminated CaM inhibition of RyR2 at Ca2+ concentrations below and above 1 μm. Five RyR2 single or double mutants in the CaM binding region (W3587A, L3591D, F3603A, W3587A/L3591D, L3591D/F3603A) eliminated or greatly reduced [35S]CaM binding and inhibition of single channel activities by CaM depending on the Ca2+ concentration. An RyR2 mutant, which assessed the effects of 4 amino acid residues that differ between RyR1 and RyR2 in or flanking the CaM binding domain, bound [35S]CaM and was inhibited by CaM, essentially identical to wild type (WT)-RyR2. Three RyR1 mutants (W3620A, L3624D, F3636A) showed responses to CaM that differed from corresponding mutations in RyR2. The results indicate that CaM regulates RyR1 and RyR2 by binding to a single, highly conserved CaM binding site and that other RyR type-specific sites are likely responsible for the differential functional regulation of RyR1 and RyR2 by CaM.

Ruthenium Red Modifies the Cardiac and Skeletal Muscle Ca2+ Release Channels (Ryanodine Receptors) by Multiple Mechanisms

The effects of ruthenium red (RR) on the skeletal and cardiac muscle ryanodine receptors (RyRs) were studied in vesicle-Ca2+ flux, [3H]ryanodine binding, and single channel measurements. In vesicle-Ca2+flux measurements, RR was more effective in inhibiting RyRs at 0.2 μm than 20 μm free Ca2+. [3H]Ryanodine binding measurements suggested noncompetitive interactions between RR inhibition and Ca2+regulatory sites of RyRs. In symmetric 0.25 m KCl with 10–20 μm cytosolic Ca2+, cytosolic RR decreased single channel activities at positive and negative holding potentials. In close to fully activated skeletal (20 μmCa2+ + 2 mm ATP) and cardiac (200 μm Ca2+) RyRs, cytosolic RR induced a predominant subconductance at a positive but not negative holding potential. Lumenal RR induced a major subconductance in cardiac RyR at negative but not positive holding potentials and several subconductances in skeletal RyR. The RR-related subconductances of cardiac RyR showed a nonlinear voltage dependence, and more than one RR molecule appeared to be involved in their formation. Cytosolic and lumenal RR also induced subconductances in Ca2+-conducting skeletal and cardiac RyRs recorded at 0 mV holding potential. These results suggest that RR inhibits RyRs and induces subconductances by binding to cytosolic and lumenal sites of skeletal and cardiac RyRs.

Knocking Down Type 2 but Not Type 1 Calsequestrin Reduces Calcium Sequestration and Release in C2C12 Skeletal Muscle Myotubes

We examined the roles of type 1 and type 2 calsequestrins (CSQ1 and CSQ2) in stored Ca2+ release of C2C12 skeletal muscle myotubes. Transduction of C2C12 myoblasts with CSQ1 or CSQ2 small interfering RNAs effectively reduced the expression of targeted CSQ protein to near undetectable levels. As compared with control infected or CSQ1 knockdown myotubes, CSQ2 and CSQ1/CSQ2 knockdown myotubes had significantly reduced stored Ca2+ release evoked by activators of intracellular Ca2+ release channel/ryanodine receptor (10 mm caffeine, 200 μm 4-chloro-m-cresol, or 10 mm KCl). Thus, CSQ1 is not essential for effective stored Ca2+ release in C2C12 myotubes despite our in vitro studies suggesting that CSQ1 may enhance ryanodine receptor channel activity. To determine the basis of the reduced stored Ca2+ release in CSQ2 knockdown myotubes, we performed immunoblot analyses and found a significant reduction in both sarco/endoplasmic reticulum Ca2+-ATPase and skeletal muscle ryanodine receptor proteins in CSQ2 and CSQ1/CSQ2 knockdown myotubes. Moreover, these knockdown myotubes exhibited reduced Ca2+ uptake and reduced stored Ca2+ release by UTP (400 μm) that activates a different family of intracellular Ca2+ release channels (inositol 1,4,5-trisphosphate receptors). Taken together, our data suggest that knocking down CSQ2, but not CSQ1, leads to reduced Ca2+ storage and release in C2C12 myotubes.

Single Channel Properties of Heterotetrameric Mutant RyR1 Ion Channels Linked to Core Myopathies

Skeletal muscle excitation-contraction coupling involves activation of homotetrameric ryanodine receptor ion channels (RyR1s), resulting in the rapid release of Ca2+ from the sarcoplasmic reticulum. Previous work has shown that Ca2+ release is impaired by mutations in RyR1 linked to Central Core Disease and Multiple Minicore Disease. We studied the consequences of these mutations on RyR1 function, following their expression in human embryonic kidney 293 cells and incorporation in lipid bilayers. RyR1-G4898E, -G4898R, and -ΔV4926/I4927 mutants in the C-terminal pore region of RyR1 and N-terminal RyR1-R110W/L486V mutant all showed negligible Ca2+ permeation and loss of Ca2+-dependent channel activity but maintained reduced K+ conductances. Co-expression of wild type and mutant RyR1s resulted in Ca2+-dependent channel activities that exhibited intermediate Ca2+ selectivities compared with K+, which suggested the presence of tetrameric RyR1 complexes composed of wild type and mutant subunits. The number of wild-type subunits to maintain a functional heterotetrameric channel differed among the four RyR1 mutants. The results indicate that homozygous RyR1 mutations associated with core myopathies abolish or greatly reduce sarcoplasmic reticulum Ca2+ release during excitation-contraction coupling. They further suggest that in individuals, expressing wild type and mutant alleles, a substantial portion of RyR1 channels is able to release Ca2+ from sarcoplasmic reticulum.

Evidence for a role of C-terminus in Ca2+ inactivation of skeletal muscle Ca2+ release channel (ryanodine receptor)

Six chimeras of the skeletal muscle (RyR1) and cardiac muscle (RyR2) Ca2+ release channels (ryanodine receptors) previously used to identify RyR1 dihydropyridine receptor interactions [Nakai et al. (1998) J. Biol. Chem. 273, 13403] were expressed in HEK293 cells to assess their Ca2+ dependence in [3H]ryanodine binding and single channel measurements. The results indicate that the C‐terminal one‐fourth has a major role in Ca2+ activation and inactivation of RyR1. Further, our results show that replacement of RyR1 regions with corresponding RyR2 regions can result in loss and/or reduction of [3H]ryanodine binding affinity while maintaining channel activity.

Effects of 2,3-Butanedione 2-Monoxime on Ca2+ Release Channels (Ryanodine Receptors) of Cardiac and Skeletal Muscle

Abstract. Single channel and [3H]ryanodine binding measurements were performed to test for a direct functional interaction between 2,3-butanedione 2-monoxime (BDM) and the skeletal and cardiac muscle sarcoplasmic reticulum Ca2+ release channels (ryanodine receptors). Single channel measurements were carried out in symmetric 0.25 m KCl media using the planar lipid bilayer method. BDM (1–10 mm) activated suboptimally Ca2+-activated (0.5–1 μm free Ca2+) single, purified and native cardiac and skeletal release channels in a concentration-dependent manner by increasing the number of channel events without a change of single channel conductances. BDM activated the two channel isoforms when added to either side of the bilayer. At a maximally activating cytosolic Ca2+ concentration of 20 μm, BDM was without effect on the cardiac channel, whereas it inhibited skeletal channel activities with IC50≈ 2.5 mm. In agreement with single channel measurements, high-affinity [3H]ryanodine binding to the two channel isoforms was increased in a concentration-dependent manner at ≤1 μm Ca2+. BDM was without a noticeable effect at low (≤0.01 μm) Ca2+ concentrations. At 20 μm Ca2+, BDM inhibited the skeletal but not cardiac channel. These results suggest that BDM regulates the Ca2+ release channels from the sarcoplasmic reticulum of skeletal and cardiac muscle in a concentration, Ca2+ and tissue-dependent manner.

Modulation of sarcoplasmic reticulum Ca2+ release in skeletal muscle expressing ryanodine receptor impaired in regulation by calmodulin and S100A1.

In vitro, calmodulin (CaM) and S100A1 activate the skeletal muscle ryanodine receptor ion channel (RyR1) at submicromolar Ca(2+) concentrations, whereas at micromolar Ca(2+) concentrations, CaM inhibits RyR1. One amino acid substitution (RyR1-L3625D) has previously been demonstrated to impair CaM binding and regulation of RyR1. Here we show that the RyR1-L3625D substitution also abolishes S100A1 binding. To determine the physiological relevance of these findings, mutant mice were generated with the RyR1-L3625D substitution in exon 74, which encodes the CaM and S100A1 binding domain of RyR1. Homozygous mutant mice (Ryr1(D/D)) were viable and appeared normal. However, single RyR1 channel recordings from Ryr1(D/D) mice exhibited impaired activation by CaM and S100A1 and impaired CaCaM inhibition. Isolated flexor digitorum brevis muscle fibers from Ryr1(D/D) mice had depressed Ca(2+) transients when stimulated by a single action potential. However, during repetitive stimulation, the mutant fibers demonstrated greater relative summation of the Ca(2+) transients. Consistently, in vivo stimulation of tibialis anterior muscles in Ryr1(D/D) mice demonstrated reduced twitch force in response to a single action potential, but greater summation of force during high-frequency stimulation. During repetitive stimulation, Ryr1(D/D) fibers exhibited slowed inactivation of sarcoplasmic reticulum Ca(2+) release flux, consistent with increased summation of the Ca(2+) transient and contractile force. Peak Ca(2+) release flux was suppressed at all voltages in voltage-clamped Ryr1(D/D) fibers. The results suggest that the RyR1-L3625D mutation removes both an early activating effect of S100A1 and CaM and delayed suppressing effect of CaCaM on RyR1 Ca(2+) release, providing new insights into CaM and S100A1 regulation of skeletal muscle excitation-contraction coupling.

Cardiac hypertrophy associated with impaired regulation of cardiac ryanodine receptor by calmodulin and S100A1

The cardiac ryanodine receptor (RyR2) is inhibited by calmodulin (CaM) and S100A1. Simultaneous substitution of three amino acid residues (W3587A, L3591D, F3603A; RyR2ADA) in the CaM binding domain of RyR2 results in loss of CaM inhibition at submicromolar (diastolic) and micromolar (systolic) Ca²⁺, cardiac hypertrophy, and heart failure in Ryr2ADA/ADA mice. To address whether cardiac hypertrophy results from the elimination of CaM and S100A1 inhibition at diastolic or systolic Ca²⁺, a mutant mouse was generated with a single RyR2 amino acid substitution (L3591D; RyR2D). Here we report that in single-channel measurements RyR2-L3591D isolated from Ryr2D/D hearts lost CaM inhibition at diastolic Ca²⁺ only, whereas S100A1 regulation was eliminated at both diastolic and systolic Ca²⁺. In contrast to the ~2-wk life span of Ryr2ADA/ADA mice, Ryr2D/D mice lived longer than 1 yr. Six-month-old Ryr2D/D mice showed a 9% increase in heart weight-to-body weight ratio, modest changes in cardiac morphology, and a twofold increase in atrial natriuretic peptide mRNA levels compared with wild type. After 4-wk pressure overload with transverse aortic constriction, heart weight-to-body weight ratio and atrial natriuretic peptide mRNA levels increased and echocardiography showed changes in heart morphology of Ryr2D/D mice compared with sham-operated mice. Collectively, the findings indicate that the single RyR2-L3591D mutation, which distinguishes the effects of diastolic and systolic Ca²⁺, alters heart size and cardiac function to a lesser extent in Ryr2D/D mice than the triple mutation in Ryr2ADA/ADA mice. They further suggest that CaM inhibition of RyR2 at systolic Ca²⁺ is important for maintaining normal cardiac function.