Reconstitution in lipid bilayer of smooth muscle cation channels activated through a GTP-binding protein.

Reconstitution of G-protein-coupled receptor activated cation channels into the lipid bilayer was attempted with plasma membrane vesicles prepared from guinea-pig ileal smooth muscle using the purification technique previously applied to the large conductance Ca2+-dependent and ATP-sensitive K+ channels (Toro et al., 1990). Under Na+-rich conditions, incorporation of plasma membrane vesicles into the bilayer produced GTPgammaS (100 microM)-activatable channel activities that are inhibited by GDPbetaS (1 mM), sensitive to Ca2+ and enhanced by depolarization. The reversal potential and unitary conductance (tens of picosiemens) of these channels varied in a manner dependent on Na+ concentration, but not affected by Cl-. These results strongly indicate that the reconstituted channels activated by GTPgammaS belong to a class of voltage-dependent, Ca2+-sensitive cation-selective channels that are activated through a G-protein, and correspond most likely to the muscarinic receptor-activated cation channels previously identified in the same preparation. These results also suggest potential usefulness of bilayer incorporation technique to investigate the receptor-operated cation channels in smooth muscle.     

Ca2+ and voltage dependence of cardiac ryanodine receptor channel block by sphingosylphosphorylcholine

The effect of sphingosylphosphorylcholine (SPC) on the cytoplasmic Ca(2+) and voltage dependence of channel gating by cardiac ryanodine receptors (RyR) was examined in lipid bilayer experiments. Micromolar concentrations of the lysosphingolipid SPC added to cis solutions rapidly and reversibly decreased the single-channel open probability (P(o)) of reconstituted RyR channels. The SPC-induced decrease in P(o) was marked by an increase in mean closed time and burst-like channel gating. Gating kinetics during intraburst periods were unchanged from those observed in the absence of the sphingolipid, although SPC induced a long-lived closed state that appeared to explain the observed decrease in channel P(o). SPC effects were observed over a broad range of cis [Ca(2+)] but were not competitive with Ca(2+). Interestingly, the sphingolipid-induced, long-lived closed state displayed voltage-dependent kinetics, even though other channel gating kinetics were not sensitive to voltage. Assuming SPC effects represent channel blockade, these results suggest that the blocking rate is independent of voltage whereas the unblocking rate is voltage dependent. Together, these results suggest that SPC binds directly to the cytoplasmic side of the RyR protein in a location in or near the membrane dielectric, but distinct from cytoplasmic Ca(2+) binding sites on the protein.     

Quercetin as a fluorescent probe for the ryanodine receptor activity in Jurkat cells

Because channels of intracellular organelles are not directly accessible to the patch-clamp technique, the activity (open probability) of intracellular ion channels in intact cells has so far eluded direct examination. Here, we present strong evidence that the ratio F380/F440 of the quercetin-specific cellular fluorescence emitted at 540 nm upon excitation at 380/440 nm reflects the open probability of an endoplasmic reticulum Ca²⁺ release channel, the ryanodine receptor (RyR), in both intact and permeabilized Jurkat cells. The time course of the Ca²⁺ release signal induced by high levels of quercetin in intact cells and that of F380/F440 were strongly correlated. The RyR specific inhibitor, ryanodine, the RyR type 3 and 1 but not type 2 specific inhibitor, dantrolene, as well as the non-specific RyR inhibitor, ruthenium red, depressed consistently the quercetin-induced Ca²⁺ transient. Confocal microscopy confirmed that the dual fluorescent signal emitted by quercetin colocalizes with the endoplasmic reticulum, not the mitochondria. A novel regulatory mechanism was identified whereby RyR activity under physiological conditions is partially suppressed (hindered channel), whereas the channel becomes nearly fully activated after exposure to millimolar concentrations of bulk cytosolic Ca²⁺ and subsequent chelation of Ca²⁺ (rectified channel). Upon rectification, the dependence of F380/F440 on the cytosolic Ca²⁺ concentration was remarkably similar to that of the open probability of the RyR type 3, not 1 or 2, reported from bilayer experiments. So, quercetin appears to be a semi-specific fluorescent probe for the activity of ryanodine receptors, which in our Jurkat (clone E6.1) cell preparations probably reports the type 3 RyR activity.     

Rapid Microfluidic Perfusion Enabling Kinetic Studies of Lipid Ion Channels in a Bilayer Lipid Membrane Chip

There is growing recognition that lipids play key roles in ion channel physiology, both through the dynamic formation and dissolution of lipid ion channels and by indirect regulation of protein ion channels. Because existing technologies cannot rapidly modulate the local (bio)chemical conditions at artificial bilayer lipid membranes used in ion channel studies, the ability to elucidate the dynamics of these lipid–lipid and lipid–protein interactions has been limited. Here we demonstrate a microfluidic system supporting exceptionally rapid perfusion of reagents to an on-chip bilayer lipid membrane, enabling the responses of lipid ion channels to dynamic changes in membrane boundary conditions to be probed. The thermoplastic microfluidic system allows initial perfusion of reagents to the membrane in less than 1 s, and enables kinetic behaviors with time constants below 10 s to be directly measured. Application of the platform is demonstrated toward kinetic studies of ceramide, a biologically important lipid known to self-assemble into transmembrane ion channels, in response to dynamic treatments of small ions (La³⁺) and proteins (Bcl-x_L mutant). The results reveal the broader potential of the technology for studies of membrane biophysics, including lipid ion channel dynamics, lipid–protein interactions, and the regulation of protein ion channels by lipid micro domains.     

RyR1 Deficiency in Congenital Myopathies Disrupts Excitation–Contraction Coupling

In skeletal muscle, excitation–contraction (EC) coupling is the process whereby the voltage‐gated dihydropyridine receptor (DHPR) located on the transverse tubules activates calcium release from the sarcoplasmic reticulum by activating ryanodine receptor (RyR1) Ca²⁺ channels located on the terminal cisternae. This subcellular membrane specialization is necessary for proper intracellular signaling and any alterations in its architecture may lead to neuromuscular disorders. In this study, we present evidence that patients with recessive RYR1‐related congenital myopathies due to primary RyR1 deficiency also exhibit downregulation of the alfa 1 subunit of the DHPR and show disruption of the spatial organization of the EC coupling machinery. We created a cellular RyR1 knockdown model using immortalized human myoblasts transfected with RyR1 siRNA and confirm that knocking down RyR1 concomitantly downregulates not only the DHPR but also the expression of other proteins involved in EC coupling. Unexpectedly, this was paralleled by the upregulation of inositol‐1,4,5‐triphosphate receptors; functionally however, upregulation of the latter Ca²⁺ channels did not compensate for the lack of RyR1‐mediated Ca²⁺ release. These results indicate that in some patients, RyR1 deficiency concomitantly alters the expression pattern of several proteins involved in calcium homeostasis and that this may influence the manifestation of these diseases.     

Effect of nicotinic acid adenine dinucleotide phosphate on ryanodine calcium release channel in heart

Nicotinic acid adenine dinucleotide phosphate (NAADP), a molecule derived from nicotinamide adenine dinucleotide phosphate (NADP+), is a recently identified nucleotide that activates Ca2+ release from intracellular stores in invertebrate eggs and in mammalian cells. NAADP could function as an intracellular messenger for mobilizing internal Ca2+ stores, however the targets and nature of NAADP-induced Ca2+ release are unknown. We report here that NAADP (3-10 microM) induces Ca2+ release from rat heart microsomes and that NAADP (1-10 microM) activates single ryanodine receptor/calcium release channels (RyR2) from dog heart incorporated into bilayer lipid membranes. The results indicate that NAADP may play a role in cardiac excitation-contraction coupling by acting on RyR2 channels.     

Gating kinetics and ligand sensitivity modified by phosphorylation of cardiac ryanodine receptors

The effects of protein-kinase- (PKA-) dependent phosphorylation on the stationary gating kinetics of single ryanodine receptor (RyR) channels was defined. The single-channel activity from canine cardiac RyR was reconstituted into planar lipid bilayers. Exogenously applied PKA increased the single-channel open probability ( P(o)) of both native and purified cardiac RyR channels, after preincubation with ATP and Mg2+. The action of PKA on the RyR channel occurred only in the presence of ATP and adenosine 5'- O-(3-thiotriphosphate) (ATPgammaS), but not in the presence of 5'-adenylimidodiphosphate (AMP-PCP). Thus, the action of PKA requires the presence of a hydrolyzable ATP analog. PKA-induced channel activation was blocked by specific PKA inhibitors. All these results confirmed that the RyR channel can be phosphorylated by exogenous protein kinase. The gating kinetics of single RyR channels before PKA treatment were significantly altered by ATP and Mg2+ as physiological ligands. In contrast, after PKA treatment, neither ATP nor Mg2+ significantly alters the gating kinetics of these channels. PKA-dependent phosphorylation thus decreases the ATP and Mg2+ apparent sensitivity in most of the gating parameters of single RyR channels. The phosphorylated RyR channels open and close more frequently, stay open for longer, and stay closed for shorter periods. The dwell-time histograms obtained demonstrate that the phosphorylated and the dephosphorylated channels have strikingly different open and closed kinetics at physiological cytoplasmic concentrations of Mg and ATP.     

Physiological role of inward rectifier K+ channels in vascular smooth muscle cells

K⁺ channels play indispensable roles in establishing the membrane potential and in regulating the contractile tone of arterial smooth muscle cells. There are four types of K⁺ channels in arterial smooth muscle: voltage-dependent K⁺ (K_V), Ca²⁺-dependent K⁺ (BK_Ca), ATP-dependent K⁺ (K_ATP), and inward rectifier K⁺ (Kir2) channels. Comparatively few physiological studies have focused on Kir2 channels because they are present only in certain small-diameter cerebral and submucosal arterioles and in coronary arterial smooth muscle. Here, we review the characteristics and regulation of Kir2 channels in vascular arterial smooth muscle. Current knowledge of the predominant Kir2 channel subtype is Kir2.1, not Kir2.2 and 2.3. Electrophysiological measurements to determine the current–voltage relationship in arterial smooth muscle revealed inward rectification with a single-channel conductance of 21 pS. Kir2 channels were found to influence the resting tone of cerebral and coronary arteries based on the fact that barium (Ba²⁺) induces the constriction of these arteries at resting tone. Kir2 channels are also highly responsive to vasoconstrictors and vasodilators. For example, the vasoconstrictors endothelin-1 and angiotensin II inhibit Kir2 channel function by activating protein kinase C (PKC), and the vasodilator adenosine stimulates Kir2 channel function by increasing the level of cAMP, which subsequently activates protein kinase A (PKA). Certain pathological conditions such as left ventricular hypertrophy are associated with a decrease in Kir2 channel expression. Although our understanding of the physiological role and regulation of Kir2 channels is incomplete, it is believed that Kir2 channels contribute to the control of vascular tone in small-diameter vessels via various intracellular signalling pathways that regulate cell membrane potential.     

The mechanism of action of Ba2+ and TEA on single Ca2+-activated K+-channels in arterial and intestinal smooth muscle cell membranes

The interaction of Ba²⁺ and TEA with Ca²⁺-activated K⁺ channels was studied in isolated membrane patches of cells from longitudinal jejunal smooth muscle of rabbit and from guinea-pig small mesenteric artery (100 m external diameter). Ba²⁺ applied from the inside of the membrane did not reduce unit current, except at high concentrations, but channels failed to open for long periods (s). This effect became much stronger when the potential gradient was in a direction driving Ba²⁺ into the channel and was reduced by increasing K⁺ ion concentration on the outside of the membrane. These results are consistent with Ba²⁺ entering the open channel and blocking at a site most of the way through the channel bore. In contrast, TEA and procaine dose-dependently reduced unit current amplitude at all patch potentials and slightly increased mean open time. Their effects were not detectably voltage-dependent and could be explained by TEA and procaine blocking the open channel with a timecourse that was faster than the frequency response of the recording system. The lack of appreciable voltage-dependence suggests that TEA and procaine bind to a site near to the inner mouth of the channel.     

The non-excitable smooth muscle: Calcium signaling and phenotypic switching during vascular disease

Calcium (Ca(2+)) is a highly versatile second messenger that controls vascular smooth muscle cell (VSMC) contraction, proliferation, and migration. By means of Ca(2+) permeable channels, Ca(2+) pumps and channels conducting other ions such as potassium and chloride, VSMC keep intracellular Ca(2+) levels under tight control. In healthy quiescent contractile VSMC, two important components of the Ca(2+) signaling pathways that regulate VSMC contraction are the plasma membrane voltage-operated Ca(2+) channel of the high voltage-activated type (L-type) and the sarcoplasmic reticulum Ca(2+) release channel, Ryanodine Receptor (RyR). Injury to the vessel wall is accompanied by VSMC phenotype switch from a contractile quiescent to a proliferative motile phenotype (synthetic phenotype) and by alteration of many components of VSMC Ca(2+) signaling pathways. Specifically, this switch that culminates in a VSMC phenotype reminiscent of a non-excitable cell is characterized by loss of L-type channels expression and increased expression of the low voltage-activated (T-type) Ca(2+) channels and the canonical transient receptor potential (TRPC) channels. The expression levels of intracellular Ca(2+) release channels, pumps and Ca(2+)-activated proteins are also altered: the proliferative VSMC lose the RyR3 and the sarcoplasmic/endoplasmic reticulum Ca(2+) ATPase isoform 2a pump and reciprocally regulate isoforms of the ca(2+)/calmodulin-dependent protein kinase II. This review focuses on the changes in expression of Ca(2+) signaling proteins associated with VSMC proliferation both in vitro and in vivo. The physiological implications of the altered expression of these Ca(2+) signaling molecules, their contribution to VSMC dysfunction during vascular disease and their potential as targets for drug therapy will be discussed.     

Different mechanisms of lysophosphatidylcholine-induced Ca mobilization in N2a mouse and SH-SY5Y human neuroblastoma cells

In mice, lysophosphatidylcholine (LPC) was found to be a physiological substrate of neuropathy target esterase, which is also bound by organophosphates that cause a delayed neuropathy in human and some animals. However, the mechanism responsible for causing the different symptoms in mice and humans that are exposed to neuropathic organophosphates still remains unknown. In the present study, we examined and compared the effect of exogenous LPC on intracellular Ca²⁺ overload in mouse N2a and human SH-SY5Y neuroblastoma cells. LPC caused an intracellular Ca²⁺ level ([Ca²⁺]_i) increase in both N2a and SH-SY5Y cells; moreover, the amplitude was higher in N2a cells than that in SH-SY5Y cells. Preincubation of the cells with verapamil, an L-type Ca²⁺ channel blocker, did not affect the LPC-induced Ca²⁺ increase in N2a cells, verapamil inhibited the response by 23% in SH-SY5Y cells. In Ca²⁺-free medium, LPC produced a significant [Ca²⁺]_i decrease in N2a cells, while it caused 64% of total [Ca²⁺]_i increase in SH-SY5Y cells. The results of a cell viability test suggest that N2a cells were more sensitive to LPC than were SH-SY5Y cells. These data suggested that the LPC-induced [Ca²⁺]_i increase was produced in each cell line through different mechanisms. In particular, the [Ca²⁺]_i increase occurred via entry through a permeabilized membrane in N2a cells, but through L-type Ca²⁺ channels as well as by Ca²⁺ release from intracellular Ca²⁺ stores in SH-SY5Y cells. Thus, the symptomatic differences of organophosphate-induced neurotoxicity between mice and humans are probably not related to the diverse amplitudes of intracellular Ca²⁺ overload produced by LPC. Moreover, the demyelination effect induced by LPC in mice may be a consequence of its detergent effect on membranes.     

Low Ca2+-sensitive maxi-K+ channels in human cultured fibroblasts

The patch clamp technique was used to reveal single channel activity in the membrane of human cultured fibroblasts. The most frequently detected ion channel type was a Ca²⁺-dependent K⁺ channel with a conductance of 287±38 pS in symmetrical 130 mM KCl. The channel showed a peculiar low Ca²⁺-sensitivity compared to that of similar channels in other preparations. In fact micromolar values of internal Ca²⁺ were not effective in the channel activation, except at high depolarizing membrane potentials. The activity was highly increased only when the channel was exposed to relatively high internal Ca²⁺ concentrations (0.2–2.0 mM).     

Variations of membrane cholesterol alter the kinetics of Ca2+-dependent K+ channels and membrane fluidity in vascular smooth muscle cells

The patch-clamp technique and fluorescence polarization analysis were used to study the dependence of Ca²⁺-dependent K⁺ channel kinetics and membrane fluidity on cholesterol (CHS) levels in the plasma membranes of cultured smooth muscle rabbit aortic cells. Mevinolin (MEV), a potent inhibitor of endogenous CHS biosynthesis was used to deplete the CHS content. Elevation of CHS concentration in the membrane was achieved using a CHS-enriching medium. Treatment of smooth muscle cells with MEV led to a nearly twofold increase in the rotational diffusion coefficient of DPH (D) and to about a ninefold elevation of probability of the channels being open (P _o). The addition of CHS to the cells membrane resulted in a nearly twofold decrease in D and about a twofold decrease in P _o. Elementary conductance of the channels did not change under these conditions. These data suggest that variations of the CHS content in the plasma membrane of smooth muscle cells affect the kinetic properties of Ca²⁺-dependent K⁺ channels presumably due to changes in plasma membrane fluidity. Our results give a possible explanation for the reported variability of Ca²⁺-dependent K⁺ channels kinetics in different preparations.     

Molecular basis of mechanotransduction in living cells

The simplest cell-like structure, the lipid bilayer vesicle, can respond to mechanical deformation by elastic membrane dilation/thinning and curvature changes. When a protein is inserted in the lipid bilayer, an energetic cost may arise because of hydrophobic mismatch between the protein and bilayer. Localized changes in bilayer thickness and curvature may compensate for this mismatch. The peptides alamethicin and gramicidin and the bacterial membrane protein MscL form mechanically gated (MG) channels when inserted in lipid bilayers. Their mechanosensitivity may arise because channel opening is associated with a change in the protein's membrane-occupied area, its hydrophobic mismatch with the bilayer, excluded water volume, or a combination of these effects. As a consequence, bilayer dilation/thinning or changes in local membrane curvature may shift the equilibrium between channel conformations. Recent evidence indicates that MG channels in specific animal cell types (e.g., Xenopus oocytes) are also gated directly by bilayer tension. However, animal cells lack the rigid cell wall that protects bacteria and plants cells from excessive expansion of their bilayer. Instead, a cortical cytoskeleton (CSK) provides a structural framework that allows the animal cell to maintain a stable excess membrane area (i.e., for its volume occupied by a sphere) in the form of membrane folds, ruffles, and microvilli. This excess membrane provides an immediate membrane reserve that may protect the bilayer from sudden changes in bilayer tension. Contractile elements within the CSK may locally slacken or tighten bilayer tension to regulate mechanosensitivity, whereas membrane blebbing and tight seal patch formation, by using up membrane reserves, may increase membrane mechanosensitivity. In specific cases, extracellular and/or CSK proteins (i.e., tethers) may transmit mechanical forces to the process (e.g., hair cell MG channels, MS intracellular Ca(2+) release, and transmitter release) without increasing tension in the lipid bilayer.     

Alterations in membrane caveolae and BKCa channel activity in skin fibroblasts in Smith-Lemli-Opitz syndrome

The Smith–Lemli–Opitz syndrome (SLOS) is an inherited disorder of cholesterol synthesis caused by mutations in DHCR7 which encodes the final enzyme in the cholesterol synthesis pathway. The immediate precursor to cholesterol synthesis, 7-dehydrocholesterol (7-DHC) accumulates in the plasma and cells of SLOS patients which has led to the idea that the accumulation of abnormal sterols and/or reduction in cholesterol underlies the phenotypic abnormalities of SLOS. We tested the hypothesis that 7-DHC accumulates in membrane caveolae where it disturbs caveolar bilayer structure–function. Membrane caveolae from skin fibroblasts obtained from SLOS patients were isolated and found to accumulate 7-DHC. In caveolar-like model membranes containing 7-DHC, subtle, but complex alterations in intermolecular packing, lipid order and membrane width were observed. In addition, the BK_Ca K⁺ channel, which co-migrates with caveolin-1 in a membrane fraction enriched with cholesterol, was impaired in SLOS cells as reflected by reduced single channel conductance and a 50 mV rightward shift in the channel activation voltage. In addition, a marked decrease in BK_Ca protein but not mRNA expression levels was seen suggesting post-translational alterations. Accompanying these changes was a reduction in caveolin-1 protein and mRNA levels, but membrane caveolar structure was not altered. These results are consistent with the hypothesis that 7-DHC accumulation in the caveolar membrane results in defective caveolar signaling. However, additional cellular alterations beyond mere changes associated with abnormal sterols in the membrane likely contribute to the pathogenesis of SLOS.     

Characterization of an outwardly rectifying chloride channel in a human submandibular gland duct cell line (HSG)

We have used the single-channel patch-clamp technique to study ion channels in the plasma membrane of the HSG human submandibular gland duct cell line. In cell-attached and excised inside-out patches, at least six channel types were observed. When the pipette contained an isotonic KCl-rich solution and the bath an isotonic NaCl-rich solution, the predominant channel type seen in excised inside-out patches was a Cl⁻ channel with an outwardly rectifying current/voltage (I/V) relation that had a conductance of 12 pS at positive pipette potentials and 43 pS at negative pipette potentials. The channel was only seen in excised patches and its open probability was not significantly increased by membrane depolarization. The channel selectivity sequence (relative to Cl⁻) was estimated from reversal potential measurements to be: SCN⁻ (1.8)>NO ₃ ⁻ (1.4)> I⁻ (1.1) ∼ Cl⁻ (1) ∼ Br⁻ (0.8) > acetate (0.35). In inside-out patches the channel was blocked by addition of 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB) (100 μmol/l) to the bath but not by 9-anthracene carboxylic acid (9-AC) (100 μmol/l). The channel was not activated by increases in the free Ca²⁺ concentration on the cytosolic surface. This is the first report of an outwardly rectifying Cl⁻ channel in a salivary epithelium. The properties of this channel are not in accordance with the properties of the Cl⁻ conductances in the acinar or duct tissues which have been studied so far and its physiological role is unclear.     

Trifluoperazine: a rynodine receptor agonist

Trifluoperazine (TFP), a phenothiazine, is a commonly used antipsychotic drug whose therapeutic effects are attributed to its central anti-adrenergic and anti-dopaminergic actions. However, TFP is also a calmodulin (CaM) antagonist and alters the Ca²⁺ binding properties of calsequestrin (CSQ). The CaM and CSQ proteins are known modulators of sarcoplasmic reticulum (SR) Ca²⁺ release in ventricular myocytes. We explored TFP actions on cardiac SR Ca²⁺ release in cells and single type-2 ryanodine receptor (RyR2) channel activity in bilayers. In intact and permeabilized ventricular myocytes, TFP produced an initial activation of RyR2-mediated SR Ca²⁺ release and over time depleted SR Ca²⁺ content. At the single channel level, TFP or nortryptiline (NRT; a tricyclic antidepressant also known to modify CSQ Ca²⁺ binding) increased the open probability (Po) of CSQ-free channels with an EC₅₀ of 5.2 μM or 8.9 μM (respectively). This Po increase was due to elevated open event frequency at low drug concentrations while longer mean open events sustained Po at higher drug concentrations. Activation of RyR2 by TFP occurred in the presence or absence of CaM. TFP may also inhibit SR Ca uptake as well as increase RyR2 opening. Our results suggest TFP and NRT can alter RyR2 function by interacting with the channel protein directly, independent of its actions on CSQ or CaM. This direct action may contribute to the clinical adverse cardiac side effects associated with these drugs.     

心房颤动时心房肌细胞L型Ca2+通道与肌浆网间Ca2+信号转导的探讨

目的：探讨房颤时心房肌细胞膜上L型Ca2+通道与肌浆网之间的Ca2+信号转导。 方法： 杂种犬10条，随机分为正常对照组和单纯房颤组。房颤组用起搏器行右心房快速起搏（500±20）次/分，术后观测24周。正常对照组不植入起搏器。胶原酶Ⅱ型分离心房肌细胞，用激光共聚焦显微镜检测L型Ca2+ 通道对细胞内Ca2+浓度变化的影响；L型Ca2+通道与肌浆网三磷酸肌醇受体（IP3R）和兰尼碱受体（RyR）之间的Ca2+信号转导。 结果： （1）L型Ca2+通道与肌浆网IP3R之间的Ca2+信号转导：正常对照组、单纯房颤组的心房肌细胞在用mibefradil和丁卡因分别阻滞T型Ca2+通道和RyR后给予细胞膜激动剂时，细胞内Ca2+浓度均升高（分别为1.4000±0.0776和1.5169±0.4414），组间比较无显著差异（P＞0.05）；（2）L型Ca2+通道与肌浆网RyR之间的Ca2+信号转导：正常对照组的心房肌细胞在用mibefradil和肝素分别阻滞T型Ca2+通道和IP3R后给予细胞膜激动剂时，细胞内Ca2+浓度升高（1.5576±0.1989），单纯房颤组的细胞内Ca2+浓度也升高（1.5372±0.2952），两组间比较无显著差异（P＞0.05）。 结论： 房颤时L型Ca2+通道与RyR和IP3R之间可能存在信号转导，但其可能在房颤时的细胞内Ca2+超载及异常Ca2+信号转导方面不起重要作用。     

Ischemic poison lysophosphatidylcholine modifies heart sodium channels gating inducing long-lasting bursts of openings

The effects of lysophosphatidylcholine (LPC) on Na channels in inside-out patches of adult rat ventricular cells using the patch-clamp technique have been investigated. Application of LPC (9-25 microM) from the inner side of membrane for 4-15 min caused a reduction of averaged Na current (INa) peak and prolonged the time course of inactivation in the potential range of -50 to -10 mV. Analysis of single channel behaviour revealed that after 30-50 min of exposure, in addition to normally functioning Na channels with short openings, LPC induced long-lasting bursts of Na channel openings (up to the 300 ms duration of the test pulses). This resulted in an appearance of noninactivated component of INa. The slope conductance of these modified channels remained the same as in control (11.3 pS - control; 11.6 pS - LPC-treated). The dwell time for modified channels increased significantly.     

Malignant hyperthermia and excitation–contraction coupling

Malignant hyperthermia (MH) is a state of elevated skeletal muscle metabolism that may occur during general anaesthesia in genetically pre‐disposed individuals. Malignant hyperthermia results from altered control of sarcoplasmic reticulum (SR) Ca²⁺ release. Mutations have been identified in MH‐susceptible (MHS) individuals in two key proteins of excitation–contraction (EC) coupling, the Ca²⁺ release channel of the SR, ryanodine receptor type 1 (RyR1) and the α1‐subunit of the dihydropyridine receptor (DHPR, L‐type Ca²⁺ channel). During EC coupling, the DHPR senses the plasma membrane depolarization and transmits the information to the ryanodine receptor (RyR). As a consequence, Ca²⁺ is released from the terminal cisternae of the SR. One of the human MH‐mutations of RyR1 (Arg614Cys) is also found at the homologous location in the RyR of swine (Arg615Cys). This animal model permits the investigation of physiological consequences of the homozygously expressed mutant release channel. Of particular interest is the question of whether voltage‐controlled release of Ca²⁺ is altered by MH‐mutations in the absence of MH‐triggering substances. This question has recently been addressed in this laboratory by studying Ca²⁺ release under voltage clamp conditions in both isolated human skeletal muscle fibres and porcine myotubes.