Mechanism of inhibition by alloxan of ATP-driven calcium transport by vascular smooth muscle microsomes

The directin vitro effects of alloxan on the Ca²⁺ handling by microsomal membranes isolated from dog mesenteric arteries were investigated. Preincubation of the vascular muscle microsomal membranes with alloxan showed a suppressive effect on both binding of Ca²⁺ (in the absence of ATP) and ATP-driven Ca²⁺ transport. Such an inhibition was time dependent, dose dependent, and temperature dependent. ATP-driven Ca²⁺ transport was much more susceptible to the inhibitory action of alloxan than Ca²⁺ binding under all experimental conditions examined. Alloxan inhibited ATP-driven Ca²⁺ transport at a comparable level over the entire period of Ca²⁺ uptake, but had no significant effect on the efflux of Ca²⁺ from preloaded microsomal membranes. This suggests that alloxan exerts its inhibitory effect on the ATP-driven Ca²⁺ transport via its action on the Ca-pump protein rather than the membrane permeability to Ca²⁺. Catalase and mannitol but not superoxide dismutase partially protected against such as inhibition by alloxan. The possible involvement of H₂O₂ mediating the inhibitory action of alloxan was further supported by the finding of a similarin vitro inhibitory effect of H₂O₂ on the ATP-driven Ca²⁺ transport by the vascular smooth muscle microsomes.     

Maltobionic acid enhances intestinal absorption of calcium and magnesium in rats

ABSTRACT

In Experiment 1, the effects of calcium maltobionate (MBCa) on calcium and magnesium absorption were examined using male rats. Four diets were designed in which 25%, 50%, and 100% of calcium carbonate (CaCO₃, Control) were substituted with MBCa and were designated as MBCa-25, MBCa-50, and MBCa-100, respectively. The cecal concentration of short-chain fatty acids was significantly higher in groups MBCa-50 and MBCa-100; however, pH of cecal contents did not significantly differ among the groups. Retention rates of calcium and magnesium were significantly higher in all MBCa groups as compared to the Control. In Experiment 2, the efficiency of calcium absorption was compared using everted sacs of jejunum and ileum with CaCO₃ and MBCa as calcium sources. More calcium from MBCa was absorbed as the concentration of calcium increased in comparison to CaCO₃. It was concluded that MBCa is a better calcium source than CaCO₃ in terms of both calcium retention and absorption.

Abbreviations: ANOVA: analysis of variance; Ca: Calcium; CaCO_3: calcium carbonate; ICP-OES: Inductivity coupled plasma optical emission spectrometer; Mg: magnesium; MBCa: calcium maltobionate; OCPC: o-cresolphthalein complexone; SCFAs: short-chain fatty acids; SE: standard error; TRPM6: transient receptor potential melastatin 6.     

Sodium-calcium exchange in mammalian heart: current-voltage relation and intracellular calcium concentration

Membrane currents and changes in intracellular calcium ion concentration ([Ca²⁺]_i) have been recorded that can be attributed to the operation of an electrogenic, voltage-dependent sodium-calcium (Na-Ca) exchanger in mammalian heart cells. Single guinea-pig ventricular myocytes under voltage clamp were perfused internally with the fluorescent Ca²⁺-indicator, fura-2, and changes in [Ca²⁺]_i and membrane current that resulted from Na-Ca exchange were isolated through the use of various organic channel blockers (verapamil, TTX), impermeant ions (Cs⁺, Ni²⁺), and inhibitors of sarcoplasmic reticulum (ryanodine). The I-V relation of Na-Ca exchange was obtained from the Ni²⁺-sensitive current elicited by ramp repolarization from +90 mV to –80 mV. Ramps were sufficiently rapid that little change in [Ca²⁺]_i occured during the ramp. The (constant) [Ca²⁺]_i during the ramp was varied over the range 100 nM to 1000 nM by varying the amplitude and duration of a pre-pulse to the ramp. The reversal potential of the Ni²⁺-sensitive ramp current varied linearly with 1n([Ca²⁺])_i. The I-V relations at different [Ca²⁺]_i over the range –60 mV to +140 mV were in reasonable accord with the predictions of a simple, simultaneous scheme of Na-Ca exchange, on the basis that only [Ca²⁺]_i had changed. The relationship between [Ca²⁺]_i and current at a constant membrane voltage was also in accord with this scheme. We suggest that Ca²⁺-fluxes through the exchanger during the cardiac action potential can be understood quantitatively by considering the binding of Ca²⁺ to the exchanger during the [Ca²⁺]_i-transient and the effects of membrane voltage on the exchanger.     

Epithelial sodium channel inhibition by AMP-activated protein kinase in oocytes and polarized renal epithelial cells

The epithelial Na(+) channel (ENaC) regulates epithelial salt and water reabsorption, processes that require significant expenditure of cellular energy. To test whether the ubiquitous metabolic sensor AMP-activated kinase (AMPK) regulates ENaC, we examined the effects of AMPK activation on amiloride-sensitive currents in Xenopus oocytes and polarized mouse collecting duct mpkCCD(c14) cells. Microinjection of oocytes expressing mouse ENaC (mENaC) with either active AMPK protein or an AMPK activator inhibited mENaC currents relative to controls as measured by two-electrode voltage-clamp studies. Similarly, pharmacological AMPK activation or overexpression of an activating AMPK mutant in mpkCCD(c14) cells inhibited amiloride-sensitive short circuit currents. Expression of a degenerin mutant beta-mENaC subunit (S518K) along with wild type alpha and gamma increased the channel open probability (P(o)) to approximately 1. However, AMPK activation inhibited currents similarly with expression of either degenerin mutant or wild type mENaC. Single channel recordings under these conditions demonstrated that neither P(o) nor channel conductance was affected by AMPK activation. Moreover, expression of a Liddle's syndrome-type beta-mENaC mutant (Y618A) greatly enhanced ENaC whole cell currents relative to wild type ENaC controls and prevented AMPK-dependent inhibition. These findings indicate that AMPK-dependent ENaC inhibition is mediated through a decrease in the number of active channels at the plasma membrane (N), presumably through enhanced Nedd4-2-dependent ENaC endocytosis. The AMPK-ENaC interaction appears to be indirect; AMPK did not bind ENaC in cells, as assessed by in vivo pull-down assays, nor did it phosphorylate ENaC in vitro. In summary, these results suggest a novel mechanism for coupling ENaC activity and renal Na(+) handling to cellular metabolic status through AMPK, which may help prevent cellular Na(+) loading under hypoxic or ischemic conditions.     

The influence of harmaline on the movements of sodium ions in smooth muscle of the guinea pig ileum

Summary The effect of changing the extracellular concentration of both Na⁺ and K⁺ on the longitudinal muscle of the guinea-pig ileum was studied in the presence and absence of harmaline. A decrease in extracellular Na⁺ concentration was found to produce a dose-dependent contractile response, which may suggest the existence of a Na...Ca exchange mechanism in this muscle. Harmaline (2 × 10⁻⁴ M) was found to reversibly inhibit this contraction and was also found to selectively block the tonic component of high-K induced contradictions. In view of the fact that harmaline is a non-competitive inhibitor of Ca-induced contractions (Hider et al., Europ. J. Pharmacol., 71, 87, 1981), the action of harmaline was interpreted as being a specific inhibitor of the Na... exchange mechanism, binding specifically to Na⁺ coordination sites.     

Regulation of cation channels in cardiac and smooth muscle cells by intracellular magnesium

Magnesium regulates various ion channels in many tissues, including those of the cardiovascular system. General mechanisms by which intracellular Mg(2+) (Mg(i)(2+)) regulates channels are presented. These involve either a direct interaction with the channel, or an indirect modification of channel function via other proteins, such as enzymes or G proteins, or via membrane surface charges and phospholipids. To provide an insight into the role of Mg(i)(2+) in the cardiovascular system, effects of Mg(i)(2+) on major channels in cardiac and smooth muscle cells and the underlying mechanisms are then reviewed. Although Mg(i)(2+) concentrations are known to be stable, conditions under which they may change exist, such as following stimulation of beta-adrenergic receptors and of insulin receptors, or during pathophysiological conditions such as ischemia, heart failure or hypertension. Modifications of cardiovascular electrical or mechanical function, possibly resulting in arrhythmias or hypertension, may result from such changes of Mg(i)(2+) and their effects on cation channels.     

Aluminium accumulation in root cell walls coincides with inhibition of root growth but not with inhibition of magnesium uptake in Norway spruce

High levels of aluminium in the soil solution of forest soils cause stress to forest trees. Within the soil profile, pH and aluminium concentration in the soil solution vary considerably with soil depth. pH strongly influences the speciation of A1 in solution, and is a factor when considering toxicity of A1 to roots. Norway spruce ( Picea abies [L.] Karst.) seedlings were grown for 7 weeks in nutrient solutions at pH 3.2, 4.0 or 5.0 containing 0, 100 or 400 µ M A1. At the end of this period, seedling growth, the cation exchange capacity of the roots and the amount of exchangeable Ca and Mg in roots were determined. A1 concentrations in whole roots, root segments, and in needles were measured. Using X‐ray microanalysis, the concentrations of Al, Ca, Mg and P were determined in cortical cell walls. We wanted to test the hypotheses that (1) the amount of Al bound to cation exchange sites can be used as a marker for Al toxicity and (2) the Mg concentration of needles is controlled by the amount of Mg bound to cation exchange sites. Low pH reduced the inhibition of Al on root growth and shoot length. Both low pH and Al lowered the concentration of Ca and Mg in needles. Al concentrations in the roots decreased as the pH decreased. In the roots, Al displaced Mg and Ca from binding sites at the root cortical cell walls. A comparison of the effects of Al at the different pH values on root growth and Mg concentration in the needles, suggests that, at pH 5.0, an Al fraction in the apoplast inhibits root growth, but does not affect Mg uptake. This fraction of Al is not available for transport to the shoots. In contrast, Mg uptake is strongly affected by Al at pH 3.2, although only very low levels of Al were detected in the roots. Thus, Al accumulation in the apoplast is a positive marker for Al effects on root growth, but not Mg uptake. The Mg concentration of needles is not controlled by the amount of Mg bound to cation exchange sites.     

Yeast mitochondrial calcium uptake: Regulation by polyamines and magnesium ions

Spermine, spermidine, and magnesium ions modulate the kinetic parameters of the Ca²⁺ transport system ofEndomyces magnusii mitochondria. Mg²⁺ at concentrations up to 5 mM partially inhibits Ca²⁺ transport with a half-maximal inhibiting concentration of 0.5 mM. In the presence of 2 mM MgCl₂, theS _0.5 value of the Ca²⁺ transport system increases from 220 to 490 µM, which indicates decreased affinity for the system. Spermine and spermidine exert an activating effect, having half-maximal concentrations of 12 and 50 µM, respectively. In the case of spermine, theS _0.5 value falls to 50–65 µM, which implies an increase in the transport system affinity for Ca²⁺. Both Mg²⁺ and spermine cause a decrease of the Hill coefficient, giving evidence for a smaller degree of cooperativity. Spermine and spermidine enable yeast mitochondria to remove Ca²⁺ from the media completely. In contrast, Mg²⁺ lowers the mitochondrial buffer capacity. When both Mg²⁺ and spermine are present in the medium, their effects on theS _0.5 value and the free extramitochondrial Ca²⁺ concentration are additive. The ability of spermine and Mg²⁺ to regulate yeast mitochondrial Ca²⁺ transport is discussed.     

Statins inhibit in vitro calcification of human vascular smooth muscle cells induced by inflammatory mediators

Although lipid-lowering therapy with 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors (statins) decreases the progression of coronary artery and aortic valve calcification, the mechanism of action of these drugs to inhibit the calcification process remains unclear. In this study, we investigated the effect of statins such as cerivastatin and atorvastatin on vascular calcification by utilizing an in vitro model of inflammatory vascular calcification. Cerivastatin and atorvastatin dose-dependently inhibited in vitro calcification of human vascular smooth muscle cells (HVSMCs) induced by the following inflammatory mediators (IM): interferon-gamma, 1alpha,25-dihydroxyvitamin D3, tumor necrosis factor-alpha, and oncostatin M. These statins also depressed expression of alkaline phosphatase (ALP) in HVSMCs induced by these factors. Mevalonate and geranylgeranylpyrophosphate reversed the inhibitory effect of cerivastatin on ALP expression in HVSMCs, while farnesylpyrophosphate showed no effect on the ALP activities inhibited by this drug, suggesting that inhibition of Rho and its downstream target, Rho kinase may mediate the inhibitory effect of cerivastatin. Cerivastatin prevented RhoA activation in HVSMCs induced by the IM. A specific inhibitor of Rho kinase (Y-27632) inhibited in vitro calcification and induction of ALP in HVSMCs. These findings provide a possible mechanism of statins to prevent the progression of calcification in inflammatory vascular diseases such as atherosclerosis and cardiac valvular calcification.     

Possible involvement of protein kinase C and cyclic AMP-dependent protein kinase in the sodium fluoride-mediated inhibition of cyclic nucleotide accumulation in smooth muscle cells

Treatment of rat thoracic aortic smooth muscle cells (A-10) with sodium fluoride (NaF) resulted in inhibition of β-adrenergic agonist—and forskolin-induced cAMP and ANF-induced cGMP accumulation and stimulation of diacylglycerol (DAG) accumulation. The concentration of NaF and treatment times required to mediate these inhibitory effects were similar to those observed for stimulation of DAG accumulation. Treatment of the cells with NaF also resulted in a loss of [³H]phorbol dibutyrate (PDBu) binding in the cytosolic portion of the cells. In addition, pre-treatment of the cells with NaF resulted in an increase in the adenylate cyclase activity. Pertussis toxin (PT) pre-treatment of the cells did not significantly affect NaF-mediated effects. Pre-treatment of the cells with protein kinase C (PKC) inhibitor staurosporin partially reversed NaF-mediated inhibition of cyclic nucleotides accumulation. These data suggest that inhibition of the formation of agonist-induced cyclic nucleotides by NaF may be due to the formation of DAG and cAMP which lead to the activation of PKC and cAMP-PK, resulting in phosphorylation of key regulatory protein(s) in the cyclic nucleotides pathway.     

Vasomotion dynamics following calcium spiking depend on both cell signalling and limited constriction velocity in rat mesenteric small arteries

Vascular smooth muscle cell contraction depends on intracellular calcium. However, calcium-contraction coupling involves a complex array of intracellular processes. Quantitating the dynamical relation between calcium perturbations and resulting changes in tone may help identifying these processes. We hypothesized that in small arteries accurate quantitation can be achieved during rhythmic vasomotion, and questioned whether these dynamics depend on intracellular signalling or physical vasoconstriction. We studied calcium-constriction dynamics in cannulated and pressurized rat mesenteric small arteries ( approximately 300 microm in diameter). Combined application of tetra-ethyl ammonium (TEA) and BayK8644 induced rhythmicity, consisting of regular and irregular calcium spiking and superposition of spikes. Calcium spikes induced delayed vasomotion cycles. Their dynamic relation could be fitted by a linear second-order model. The dirac impulse response of this model had an amplitude that was strongly reduced with increasing perfusion pressure between 17 and 98 mmHg, while time to peak and relaxation time were the largest at an intermediate pressure (57 mmHg: respectively 0.9 and 2.3 sec). To address to what extent these dynamics reside in intracellular signalling or vasoconstriction, we applied rhythmic increases in pressure counteracting the vasoconstriction. This revealed that calcium-activation coupling became faster when vasoconstriction was counteracted. During such compensation, a calcium impulse response remained that lasted 0.5 sec to peak activation, followed by a 1.0 sec relaxation time, attributable to signalling dynamics. In conclusion, this study demonstrates the feasibility of quantitating calcium-activation dynamics in vasomoting small arteries. These dynamics relate to both intracellular signalling and actual vasoconstriction. Performing such analyses during pharmacological intervention and in genetic models provides a tool for unravelling calcium-contraction coupling in small arteries.     

不同牵张刺激对豚鼠胃窦环行肌细胞电压依赖性钙电流的影响

利用全细胞膜片钳技术,在胃窦环行肌细胞上观察了不同方式的牵张刺激对电压依赖性钙电流的影响,探讨牵张刺激对胃窦平滑肌细胞电压依赖性钙电流的作用。用低渗性溶液灌流细胞引起的牵张刺激首先增加电压依赖性钙电流,接着激活一种内向性钳制电流。钙电流的增加发生在灌流后１ｍｉｎ内,而内向性钳制电流在细胞明显膨胀之后缓慢激活。低渗和正压引起的细胞膨胀明显增加电压依赖性钙离子电流,而利用两个电极直接牵细胞则不出现钙电     

Effects of zinc,selenium, and calcium on the nephrotoxicity of cadmium in primary cultures of rat renal proximal epithelial cells

The influence of essential metals, like zinc, selenium, and calcium, on the nephrotoxicity of cadmium was studied in primary cultures of rat proximal tubular cells. Damage to kidney cells was assessed by measuring the release of lactate dehydrogenase (LDH), γ-glutamyltranspeptidase (GTP), and β-N-acetylglucosaminidase (NAG) from cells into the medium and the cellular concentration of protein. Incubation with 200 μM cadmium in the presence of equivalent molar or lower concentrations of zinc and selenium showed greater release of LDH and NAG than cadmium alone, indicating an enhanced effect. However, metallothionein (MT) induced by pretreatment with a nontoxic concentration of zinc decreased significantly the release of enzyme from cells and elevated cellular protein levels in response to MT levels. MT provided partial protection against the nephrotoxicity of cadmium. Decreased calcium levels in the incubation medium also resulted in markedly increased release of LDH and NAG from cells exposed to cadmium and reduced cellular protein levels. These findings suggest that variations in zinc and calcium intake may affect the development of cadmium-induced renal dysfunction.     

Dependence of alignment direction on magnitude of strain in esophageal smooth muscle cells

The response of cells in vitro to mechanical forces has been the subject of much research using devices to exert controlled mechanical stimulation on cultured cells or isolated tissue. In this study, esophageal smooth muscle cells were seeded on flexible polyurethane membranes to form a confluent cell layer. The cells were then subjected to uniform cyclic stretch of varying magnitudes at a frequency of approximately five cycles per minute in a custom made mechatronic bioreactor, providing similar strains experienced in the in vivo mechanical environment of the esophagus. The results show that the orientation response is dependent on the magnitude of cyclic stretch applied. Smooth muscle cells showed parallel alignment to the force direction at low cyclic strains (2%) compared to the hill‐valley morphology of static controls. At higher strains (5% and 10% magnitude), the cells exhibited a consistent alignment perpendicular to the strain. To our knowledge, this is the first time that the alignment direction's dependence on strain magnitude has been demonstrated. MTS analysis indicated that cell metabolism was reduced when mechanical strain was applied, and proliferation was inhibited by mechanical strain. Protein expression indicates a decrease in smooth muscle α‐actin, indicative of changes in cell phenotype, an increase in vimentin, which is associated with increased cell motility, and an increase in desmin, indicating differentiation in stimulated cells. Biotechnol. Bioeng. 2009;102: 1703–1711. © 2008 Wiley Periodicals, Inc.     

Imipramine inhibition of TRPM‐like plasmalemmal Mg2+ transport in vascular smooth muscle cells

Depression is associated with vascular disease, such as myocardial infarction and stroke. Pharmacological treatments may contribute to this association. On the other hand, Mg²⁺ deficiency is also known to be a risk factor for the same category of diseases. In the present study, we examined the effect of imipramine on Mg²⁺ homeostasis in vascular smooth muscle, especially via melastatin‐type transient receptor potential (TRPM)‐like Mg²⁺‐permeable channels. The intracellular free Mg²⁺ concentration ([Mg²⁺]_i) was measured using ³¹P‐nuclear magnetic resonance (NMR) in porcine carotid arteries that express both TRPM6 and TRPM7, the latter being predominant. pH_i and intracellular phosphorus compounds were simultaneously monitored. To rule out Na⁺‐dependent Mg²⁺ transport, and to facilitate the activity of Mg²⁺‐permeable channels, experiments were carried out in the absence of Na⁺ and Ca²⁺. Changing the extracellular Mg²⁺ concentration to 0 and 6 mM significantly decreased and increased [Mg²⁺]_i, respectively, in a time‐dependent manner. Imipramine statistically significantly attenuated both of the bi‐directional [Mg²⁺]_i changes under the Na⁺‐ and Ca²⁺‐free conditions. This inhibitory effect was comparable in influx, and much more potent in efflux to that of 2‐aminoethoxydiphenyl borate, a well‐known blocker of TRPM7, a channel that plays a major role in cellular Mg²⁺ homeostasis. Neither [ATP]_i nor pH_i correlated with changes in [Mg²⁺]_i. The results indicate that imipramine suppresses Mg²⁺‐permeable channels presumably through a direct effect on the channel domain. This inhibitory effect appears to contribute, at least partially, to the link between antidepressants and the risk of vascular diseases.     

Hyperosmolality-induced abnormal patterns of calcium mobilization in smooth muscle cells from non-diabetic and diabetic rats

Hyperglycemia and/or hyperosmolality may disturb calcium homeostasis in vascular smooth muscle cells (SMCs), leading to altered vascular contractility in diabetes. To test this hypothesis, the KCl induced increases in [Ca²⁺]_i in primarily cultured vascular SMCs exposed to different concentrations of glucose were examined. With glucose concentration in solutions kept at 5.5 mM, KCl induced a fast increase in [Ca²⁺]_i which then slowly declined (type 1 response) in 83% of SMCs from non-diabetic rats. In 9% of non-diabetic SMCs KCl induced a slow increase in [Ca²⁺]_i (type 2 response). Interestingly, under the same culture conditions KCl induced type 1 and type 2 responses in 47 and 35% of SMCs from diabetic rats. When SMCs from non-diabetic or diabetic rats were cultured in 36 mM glucose, KCl induced a fast increase in [Ca²⁺]_i which, however, maintained at a high level (type 3 response). The sustained level of [Ca²⁺]_i in the presence of KCl was significantly higher in cells cultured with 36 mM glucose than that in non-diabetic cells cultured with 5.5 mM glucose. Furthermore, the hyperglycemia-induced alterations in calcium mobilization were similarly observed in cells cultured in high concentration of mannitol (30.5 mM) or L-glucose, indicating that hyperosmolality was mainly responsible for the abnormal calcium mobilization in diabetic SMCs.     

Hypertriglyceridemic serum from magnesium-deficient rats induces proliferation and lipid accumulation in cultured vascular smooth muscle cells

An important characteristic of hyperlipemia associated with magnesium deficiency in rats is the postprandial accumulation of triglyceride-rich lipoproteins. The present investigation was performed to determine the effect of serum from magnesium-deficient animals on cultured vascular smooth muscle cells (VSMC). Sera were obtained from control and magnesium-deficient rats fed adequate or deficient diets for 8 days. Magnesium-deficient animals were hypertriglyceridemic compared with control rats, but their total cholesterolemia was not significantly modified. Pooled sera from control and magnesium-deficient animals were added to the culture medium at various concentrations. The maximum of proliferation for both control and magnesium-deficient sera was reached when they were added at 6% to the culture medium and on day 4 after the begining of incubation. Medium containing serum from magnesium-deficient rats stimulated the cell proliferation as monitored by cell count and [³H]thymidine incorporation. Staining of VSMC with Oil red O and measuring lipids have shown a marked lipid accumulation (triglycerides) in cells incubated with serum obtained from magnesium-deficient animals compared with serum from control rats. These results indicate that serum from magnesium-deficient rats contains factors that stimulate proliferation of arterial medial cells and that hyperlipemia associated with magnesium-deficiency may cause lipid accumulation in vascular cells.     

Channel Gating Dependence on Pore Lining Helix Glycine Residues in Skeletal Muscle Ryanodine Receptor

Type 1 ryanodine receptors (RyR1s) release Ca²⁺ from the sarcoplasmic reticulum to initiate skeletal muscle contraction. The role of RyR1-G4934 and -G4941 in the pore-lining helix in channel gating and ion permeation was probed by replacing them with amino acid residues of increasing side chain volume. RyR1-G4934A, -G4941A, and -G4941V mutant channels exhibited a caffeine-induced Ca²⁺ release response in HEK293 cells and bound the RyR-specific ligand [³H]ryanodine. In single channel recordings, significant differences in the number of channel events and mean open and close times were observed between WT and RyR1-G4934A and -G4941A. RyR1-G4934A had reduced K⁺ conductance and ion selectivity compared with WT. Mutations further increasing the side chain volume at these positions (G4934V and G4941I) resulted in reduced caffeine-induced Ca²⁺ release in HEK293 cells, low [³H]ryanodine binding levels, and channels that were not regulated by Ca²⁺ and did not conduct Ca²⁺ in single channel measurements. Computational predictions of the thermodynamic impact of mutations on protein stability indicated that although the G4934A mutation was tolerated, the G4934V mutation decreased protein stability by introducing clashes with neighboring amino acid residues. In similar fashion, the G4941A mutation did not introduce clashes, whereas the G4941I mutation resulted in intersubunit clashes among the mutated isoleucines. Co-expression of RyR1-WT with RyR1-G4934V or -G4941I partially restored the WT phenotype, which suggested lessening of amino acid clashes in heterotetrameric channel complexes. The results indicate that both glycines are important for RyR1 channel function by providing flexibility and minimizing amino acid clashes.