Local Ca2+ entry through L-type Ca2+ channels activates Ca2+-dependent K+ channels in rabbit coronary myocytes.

Large-conductance Ca2+-dependent K+ channels (KCa), which are abundant on the sarcolemma of vascular myocytes, provide negative feedback via membrane hyperpolarization that limits Ca2+ entry through L-type Ca2+ channels (ICaL). We hypothesize that local accumulation of subsarcolemmal Ca2+ during ICaL openings amplifies this feedback. Our goal was to demonstrate that Ca2+ entry through voltage-gated ICaL channels can stimulate adjacent KCa channels by a localized interaction in enzymatically isolated rabbit coronary arterial myocytes voltage clamped in whole-cell or in cell-attached patch clamp mode. During slow-voltage-ramp protocols, we identified an outward KCa current that is activated by a subsarcolemmal Ca2+ pool dissociated from bulk cytosolic Ca2+ pool (measured with indo 1) and is dependent on L-type Ca2+ channel activity. Transient activation of unitary KCa channels in cell-attached patches could be detected during long step depolarizations to +40 mV (holding potential, -40 mV; 219 pS in near-symmetrical K+). This local interaction between the channels required the presence of Ca2+ in the pipette solution, was enhanced by the ICaL agonist Bay K 8644, and persisted after impairment of the sarcoplasmic reticulum by incubation with 10 micromol/L ryanodine and 30 micromol/L cyclopiazonic acid for at least 60 minutes. Furthermore, we provide the first direct evidence of simultaneous openings of single KCa (67 pS) and ICaL (3.9 pS) channels in near-physiological conditions, near resting membrane potential. Our data imply a novel sensitive mechanism for regulating resting membrane potential and tone in vascular smooth muscle.     

Behavior of nonselective cation channels and large-conductance Ca2+-activated K+ channels induced by dynamic changes in membrane stretch in cultured smooth muscle cells of human coronary artery

INTRODUCTION: The effects of membrane stretch on ion channels were investigated in cultured smooth muscle cells of human coronary artery. METHODS AND RESULTS: In the cell-attached configuration, membrane stretch with negative pressure induced two types of stretch-activated (SA) ion channels: a nonselective cation channel and a large-conductance Ca2+-activated K+ (BK(Ca)) channel. The single-channel conductances of SA cation and BK(Ca) channels were 26 and 203 pS, respectively. To elucidate the mechanism of activation of these SA channels and to minimize mechanical disruption, a sinusoidal change in pipette pressure was applied to the on-cell membrane patch. During dynamic changes in pipette pressure, increases in SA cation channel activity was found to coincide with increases in BK(Ca) channel activity. In the continued presence of cyclic stretch, the activity of SA cation channels gradually diminished. However, after termination of cyclic stretch, BK(Ca) channel activity was greatly enhanced, but the activity of SA cation channels disappeared. CONCLUSION: This study is the first to demonstrate that the behavior of SA cation and BK(Ca) channels in coronary smooth muscle cells is differentially susceptible to dynamic changes in membrane tension.     

Calcitonin gene-related peptide activates the K+ channels of vascular smooth muscle cells via adenylate cyclase

The aim of the present study was to examine the effects of calcitonin gene-related peptide (CGRP) on the K⁺ channels of vascular smooth muscle cells. Cultured smooth muscle cells from a porcine coronary artery were studied using the patch-clamp technique. Extracellular application of 100 nM CGRP activated two types of K⁺ channels the Ca²⁺-activated K⁺ channel (K_Ca channel) and the ATP-sensitive K⁺ channel (K_ATP channel) in cell-attached patch configurations. In cells pretreated with Rp-cAMPS, a membrane-permeable inhibitor of cAMP-dependent protein kinase (PKA), extracellular application of 100 nM CGRP could not activate the K_Ca or K_ATP channel, indicating that the activation of the K⁺ channels by CGRP occurs in connection with PKA. In the cell-attached patch configurations, extracellular application of 1 mM dibutyryl cAMP, a membrane permeable cAMP, activated K_Ca and K_ATP channels. In inside-out patch configurations, application of PKA to the cytosolic side activated both the K_Ca and K_ATP channels. These results indicate that CGRP modulates the K⁺ channels of vascular smooth muscle cells via adenylate cyclase, i.e., cAMP-PKA pathway, and contributes to control of vascular tone.     

Ionic currents in single smooth muscle cells of the canine renal artery.

Membrane currents from single smooth muscle cells enzymatically isolated from canine renal artery were recorded using the patch-clamp technique in the whole-cell and cell-attached configurations. These cells exhibited a mean resting potential, input resistance, membrane time constant, and cell capacitance of -51.8 +/- 2.1 mV, 5.2 +/- 0.98 G omega, 116.2 +/- 16.4 msec, and 29.1 +/- 2.0 pF, respectively. Inward current, when elicited from a holding potential of -80 mV, activated near -50 mV, reached a maximum near 0 mV and was sensitive to the dihydropyridine agonist Bay K 8644 and dihydropyridine antagonist nisoldipine. Two components of macroscopic outward current were identified from voltage-step and ramp depolarizations. The predominant charge carrier of the net outward current was identified as K+ by tail-current experiments (reversal potential, -61.0 +/- 0.8 mV in 10.8 mM [K+]o 0 mM [K+]i). The first component was a small, low-noise, voltage- and time-dependent current that activated between -40 and -30 mV (IK(dr)), and the second component was a larger, noisier, voltage- and time-dependent current that activated at potentials positive to +10 mV (IK(Ca)). Both IK(dr) and IK(Ca) displayed little inactivation during long (4-second) voltage steps. IK(Ca) and IK(dr) could be pharmacologically separated by using various Ca2+ and K+ channel blockers. IK(Ca) was substantially inhibited by external NiCl2 (500 microM), CdCl2 (300 microM), EGTA (5 mM), tetraethylammonium (Ki at +60 mV, 307 microM), and charybdotoxin (100 nM) but was insensitive to 4-aminopyridine (0.1-10 mM). IK(dr) was inhibited by 4-aminopyridine (Ki at +10 mV, 723 microM) and tetraethylammonium (Ki at +10 mV, 908 microM) but was insensitive to external NiCl2 (500 microM), CdCl2 (300 microM), EGTA (5 mM), and charybdotoxin (100 nM). Two types of single K+ channels were identified in cell-attached patches. The most abundant K+ channel that was recorded exhibited voltage-dependent activation, was blocked by external tetraethylammonium (250 microM), and had a large single-channel conductance (232 +/- 12 pS with 150 mM K+ in the patch pipette, 130 +/- 17 pS with 5.4 mM K+ in the patch pipette). The second channel was also voltage dependent, was blocked by 4-aminopyridine (5 mM), and exhibited a smaller single-channel conductance (104 +/- 8 pS with 150 mM K+ in the patch pipette, 57 +/- 6 pS with 5.4 mM K+ in the patch pipette). These results suggest that depolarization of canine renal artery cells opens dihydropyridine-sensitive Ca2+ channels and at least two K+ channels.(ABSTRACT TRUNCATED AT 400 WORDS)     

Effect of adenosine triphosphate-sensitive potassium channel inhibitors on coronary metabolic vasodilation

The ATP-sensitive potassium (K(ATP)) channel is a distinct type of potassium ion channel that is found in the vascular smooth muscle cells of a variety of mammalian species, including humans. The activity of K(ATP) channels is determined by many factors including cellular ATP and ADP levels, thus providing a link between cellular metabolism and vascular tone through its effects on membrane potential. Experimental studies using inhibitors of K(ATP) channels, such as the sulfonuylurea class of drugs, indicate that these channels modulate coronary vascular tone including the hyperaemia induced by increased myocardial metabolism. This review examines the evidence linking K(ATP) channels to the regulation of coronary vascular tone and the potential clinical implications of pharmacologic therapies that act on K(ATP) channels.     

Ionic currents in single cells from human cystic artery.

The patch-clamp technique was used to study the electrophysiological properties of single smooth muscle cells obtained from the human cystic artery. These cells contracted on exposure to high K+ and had a mean resting potential of -36 +/- 7 mV. Under current clamp, regenerative responses could not be elicited when depolarizing pulses were applied. Voltage-clamp measurements demonstrated that a large fraction of the outward current was inhibited by tetraethylammonium (5-10 mM) or Ca2+ channel blockers and that it was enhanced by increasing [Ca2+]o, suggesting that it is a Ca(2+)-activated K+ current. In addition, spontaneous transient outward currents that were sensitive to extracellular Ca2+ were observed in some cells. In cell-attached patch-clamp recordings, Ca(2+)-activated K+ channels that had a conductance of 117 pS were consistently identified. At negative potentials (approximately -60 mV), these single-channel events deactivated completely and very quickly, suggesting that they do not control the resting membrane potential in healthy cystic artery cells. Ca2+ currents that were recorded using Ba2+ (10 mM) as the charge carrier were enhanced by the dihydropyridine agonist, Bay K 8644, and blocked by nifedipine (0.1 microM). Only one type of Ca2+ current, the L-type, could be identified in these cells. These results demonstrate that the major ionic currents in the human cystic artery are similar to other mammalian arteries and indicate that this tissue will be a useful model for studying the metabolic and pharmacological modulation of ionic currents in human vascular smooth muscle.     

Effects of K+ channel blockers on vascular tone in the perfused rat lung

To learn more of the role of K+ channel activity in the regulation of pulmonary vascular tone, we compared the pressor effects of the differential blockers of numerous K+ channels, tetraethylammonium chloride and 4-aminopyridine, and the inhibitor of ATP-sensitive K+ channels glibenclamide in meclofenamate-treated salt solution-perfused rat lungs. Tetraethylammonium (1 to 20 mM) and 4-aminopyridine (1 to 10 mM), but not glibenclamide (1 to 20 microM) caused vasoconstriction in the normoxic lung. The Ca++ channel blocker nifedipine (0.1 microM) and the alpha adrenoceptor antagonist phentolamine (10 microM) inhibited the 4-aminopyridine response by about 50% and reduced slightly the smaller tetraethylammonium response. 4-Aminopyridine and, to a lesser extent, tetraethylammonium, but not glibenclamide, also potentiated peak vasoconstriction to angiotensin II and airway hypoxia. Nifedipine, but not phentolamine, inhibited hypoxic vasoconstriction and prevented the potentiation by 4-aminopyridine. These results suggest that Ca(++)- and/or voltage-activated (not ATP-sensitive) K+ channels may be important in maintaining low pulmonary vascular tone.     

K+ channel openers activate brain sulfonylurea-sensitive K+ channels and block neurosecretion.

Vascular K+ channel openers such as cromakalim, nicorandil, and pinacidil potently stimulate 86Rb+ efflux from slices of substantia nigra. This 86Rb+ efflux is blocked by antidiabetic sulfonylureas, which are known to be potent and specific blockers of ATP-regulated K+ channels in pancreatic beta cells, cardiac cells, and smooth muscle cells. K0.5, the half-maximal effect of the enantiomer (-)-cromakalim, is as low as 10 nM, whereas K0.5 for nicorandil is 100 nM. These two compounds appear to have a much higher affinity for nerve cells than for smooth muscle cells. Openers of sulfonylurea-sensitive K+ channels lead to inhibition of gamma-aminobutyric acid release. There is an excellent relationship between potency to activate 86Rb+ efflux and potency to inhibit neurotransmitter release.     

Ca(2+)-activated potassium channels and ATP-sensitive potassium channels as modulators of vascular tone

Membrane hyperpolarization through activation of potassium channels in arterial smooth muscle appears to be an effective mechanism to dilate arteries. Conversely, membrane depolarization through inhibition of potassium channels can lead to vasoconstriction. Here, I briefly review the roles of Ca(2+)-activated K(+) (K(Ca)) channels and ATP-sensitive K(+) (K(ATP)) channels in the control of arterial smooth muscle function. K(Ca) channels regulate arterial tone in response to changes in intravascular pressure and possibly to a variety of vasoconstrictors. K(ATP) channels respond to changes in the cellular metabolic state and are targets of a variety of synthetic and endogenous vasodilators.     

大电导钙激活钾通道在高血压病中的研究进展

大电导钙激活钾通道（BKCa）是血管平滑肌细胞（VSMCs）上表达最丰富的钾通道,对维持VSMCs的膜电位及血管收缩和舒张的动态平衡具有重要的调节作用。BKCa通道的激活可使细胞膜发生超极化,从而抑制电压依赖性钙通道的激活和钙离子内流,导致平滑肌舒张。对高血压患者的观察和高血压动物模型的研究发现,高血压血管张力升高时平滑肌细胞膜表面钾离子和钙离子通道表达和功能均发生异常,因此,有人推测高血压是离子通道重构导致平滑肌细胞去极化的结果。本文主要综述近年来BKCa通道在高血压病中的研究进展。     

Calcium sparks in human coronary artery smooth muscle cells resolved by confocal imaging

OBJECTIVE: The observation of local 'elementary' Ca2+ release events (Ca2+ sparks) through ryanodine receptor (RyR) channels in the sarcoplasmic reticulum (SR) has changed our understanding of excitation-contraction (EC) coupling in cardiac and smooth muscle. In arterial smooth muscle, Ca2+ sparks have been suggested to oppose myogenic vasoconstriction and to influence vasorelaxation by activating co-localized Ca2+ activated K+ (K(Ca)) channels (STOCs). However, all prior studies on Ca2+ sparks have been performed in non-human tissues. METHODS: In order to understand the possible significance of Ca2+ sparks to human cardiovascular function, we used high spatial resolution confocal imaging to record Ca2+ sparks in freshly-isolated, individual myocytes of human coronary arteries loaded with the Ca2+ indicator fluo-3. RESULTS: Local SR Ca2+ release events recorded in human myocytes were similar to 'Ca2- sparks' recorded previously from non-human smooth muscle cells. In human myocytes, the peak [Ca2+]i amplitudes of Ca2+ sparks (measured as F/F0) and width at half-maximal amplitude were 2.3 and 2.27 microm, respectively. The duration of Ca2+ sparks was 62 ms. Ca2+ sparks were completely inhibited by ryanodine (10 micromol/l). Ryanodine-sensitive STOCs could be identified with typical properties of K(Ca) channels activated by Ca2+ sparks. CONCLUSION: Our data implies that modern concepts suggesting an essential role of Ca2+ spark generation in EC coupling recently derived from non-human muscle are applicable to human cardiovascular tissue. Although the basic properties of Ca2+ sparks are similar, our results demonstrate that Ca2+ sparks in coronary arteries in humans, have features distinct from non-arterial smooth muscle cells of other species.     

离子通道与缺氧性肺血管收缩

氧敏感性离子通道广泛表达于肺动脉平滑肌细胞,它们在低氧时决定血管的紧张度,参与缺氧性肺血管收缩的发生.本文就急性和慢性缺氧性肺血管收缩时钾离子和钙离子发挥的作用作一简单综述.     

Outward potassium currents in freshly isolated smooth muscle cell of dog coronary arteries

Outward membrane currents were characterized in single coronary smooth muscle cells of adult beagle dogs. The cells averaged 96.4 x 7.1 microns and had a resting potential of -50.7 mV, an input resistance of 307.9 M omega, a capacitance of 32.3 pF, and a calculated membrane surface area of 4,037 microns2. The cells contracted in response to external application of acetylcholine or high K+. In voltage clamp by use of the suction pipette method, outward current began to appear at -50 mV and reached 15.2 nA at 50 mV with a current density of 376.5 microA/cm2. The current was reduced by external tetraethylammonium, Ba2+, and internal Cs+, and its reversal potential had a Nernst relation to external K+ concentration. Elevation of external Ca2+ (Ca2+o) from 0 to 0.3 mM increased total K+ current by up to 300%; elevation of internal Ca2+ (Ca2+i) to 5 x 10(-7) M by internal perfusion increased total outward current to a similar extent, suggesting a large difference in Ca2+ transmembrane sensitivity. Total whole-cell K+ current consisted of two components: an initial time-independent current (Ii) followed by a time-dependent current (It). Ii and It were through separate K+ channels based on differences in a) sensitivity to Ca2+09b) modulation by an inward Ca2+ current, c) current amplitudes and activation kinetics, and d) responses to pharmacological agents. It was the largest component, measuring 4.5 nA in 0 mM Ca2+o but increasing to 11.9 nA in 0.3 mM Ca2+o with a steep 2.5 power function. It activated with a biexponential time course; in Ca2+o-free solution, its time course was relatively insensitive to voltage changes but became voltage sensitive in the presence of Ca2+o. Further, such sensitivity was abolished or enhanced by Co2+ or Bay K 8644, respectively. We concluded that there are two types of Ca2+-sensitive K+ currents, Ii and It, in coronary smooth muscle cells. Via an inward Ca2+ channel Ca2+o strongly modulates It, both in amplitude and kinetics.     

Vasopressin inhibits sarcolemmal ATP-sensitive K+ channels via V1 receptors activation in the guinea pig heart.

To examine the effect of vasopressin on the sarcolemmal ATP-sensitive K (K(ATP)) channel, cell-attached, insideout and open-cell-attached methods of patch clamp techniques were used in isolated guinea pig ventricular myocytes. Suppressing both glycolytic and oxidative ATP production attained K(ATP) channel activation. In the cell-attached mode, vasopressin inhibited KATP channels in a concentration-dependent manner with an IC50 of 15.1+/-1.8 nmol/L. In the inside-out configuration, vasopressin failed to block K(ATP) channels. In the cell-attached mode, manning compound (1 micromol/L), a V1 receptor-selective antagonist, blocked the inhibitory action of vasopressin, although OPC-31260 (1 micromol/L), a V2 receptor-selective antagonist could not affect the action of vasopressin. In addition, vasopressin lost its inhibitory action on K(ATP) channels when the channel was activated by pinacidil, a K channel opener and in the open-cell-attached mode effected by streptolysin-O. Thus, the inhibitory action of vasopressin K(ATP) channels may occur via V1 receptor related mechanism.     

Mechanosensitive cation channels in arterial smooth muscle cells are activated by diacylglycerol and inhibited by phospholipase C inhibitor

Mechanosensitive cation channels may be involved in the development of the myogenic tone of arteries. The molecular identity of these channels is not clear, but transient receptor potential channels (TRPCs) are good candidates. In the present study, we searched for mechanosensitive channels at the single-channel level in arterial smooth muscle cells using the patch-clamp technique and investigated the channel properties in the light of properties of TRPCs. With 140 mmol/L CsCl in the pipette solution, application of negative pressures to the back of the pipette induced the activation of channels the open probability of which increased with the amount of negative pressure. The current-voltage relationship was linear in symmetrical ionic conditions, and the single-channel conductances for Cs+, K+, and Na+ were 30, 36, and 27 pS, respectively. When NMDG+ was substituted for Cs+ in the pipette solution, inward currents were abolished, whereas outward currents remained active, indicating that the channels were nonselective to cations. The channel activity was blocked by intracellular Gd3+ and 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid and increased by diacylglycerol and by cyclopiazonic acid. Phospholipase C inhibitor (U73122) inhibited not only channel activity but also the development of myogenic tone induced by stretching of the basilar arteries. These results suggest that the ion channel responsible for the development of myogenic tone is the 30-pS mechanosensitive cation channel that exhibits properties similar to those of TRPCs.     

NO hyperpolarizes pulmonary artery smooth muscle cells and decreases the intracellular Ca2+ concentration by activating voltage-gated K+ channels.

NO causes pulmonary vasodilation in patients with pulmonary hypertension. In pulmonary arterial smooth muscle cells, the activity of voltage-gated K+ (Kv) channels controls resting membrane potential. In turn, membrane potential is an important regulator of the intracellular free calcium concentration ([Ca2+]i) and pulmonary vascular tone. We used patch clamp methods to determine whether the NO-induced pulmonary vasodilation is mediated by activation of Kv channels. Quantitative fluorescence microscopy was employed to test the effect of NO on the depolarization-induced rise in [Ca2+]i. Blockade of Kv channels by 4-aminopyridine (5 mM) depolarized pulmonary artery myocytes to threshold for initiation of Ca2+ action potentials, and thereby increased [Ca2+]i. NO (approximately 3 microM) and the NO-generating compound sodium nitroprusside (5-10 microM) opened Kv channels in rat pulmonary artery smooth muscle cells. The enhanced K+ currents then hyperpolarized the cells, and blocked Ca(2+)-dependent action potentials, thereby preventing the evoked increases in [Ca2+]i. Nitroprusside also increased the probability of Kv channel opening in excised, outside-out membrane patches. This raises the possibility that NO may act either directly on the channel protein or on a closely associated molecule rather than via soluble guanylate cyclase. In isolated pulmonary arteries, 4-aminopyridine significantly inhibited NO-induced relaxation. We conclude that NO promotes the opening of Kv channels in pulmonary arterial smooth muscle cells. The resulting membrane hyperpolarization, which lowers [Ca2+]i, is apparently one of the mechanisms by which NO induces pulmonary vasodilation.     

TRPV4 forms a novel Ca2+ signaling complex with ryanodine receptors and BKCa channels

Vasodilatory factors produced by the endothelium are critical for the maintenance of normal blood pressure and flow. We hypothesized that endothelial signals are transduced to underlying vascular smooth muscle by vanilloid transient receptor potential (TRPV) channels. TRPV4 message was detected in RNA from cerebral artery smooth muscle cells. In patch-clamp experiments using freshly isolated cerebral myocytes, outwardly rectifying whole-cell currents with properties consistent with those of expressed TRPV4 channels were evoked by the TRPV4 agonist 4alpha-phorbol 12,13-didecanoate (4alpha-PDD) (5 micromol/L) and the endothelium-derived arachidonic acid metabolite 11,12 epoxyeicosatrienoic acid (11,12 EET) (300 nmol/L). Using high-speed laser-scanning confocal microscopy, we found that 11,12 EET increased the frequency of unitary Ca2+ release events (Ca2+ sparks) via ryanodine receptors located on the sarcoplasmic reticulum of cerebral artery smooth muscle cells. EET-induced Ca2+ sparks activated nearby sarcolemmal large-conductance Ca2+-activated K+ (BKCa) channels, measured as an increase in the frequency of transient K+ currents (referred to as "spontaneous transient outward currents" [STOCs]). 11,12 EET-induced increases in Ca2+ spark and STOC frequency were inhibited by lowering external Ca2+ from 2 mmol/L to 10 micromol/L but not by voltage-dependent Ca2+ channel inhibitors, suggesting that these responses require extracellular Ca2+ influx via channels other than voltage-dependent Ca2+ channels. Antisense-mediated suppression of TRPV4 expression in intact cerebral arteries prevented 11,12 EET-induced smooth muscle hyperpolarization and vasodilation. Thus, we conclude that TRPV4 forms a novel Ca2+ signaling complex with ryanodine receptors and BKCa channels that elicits smooth muscle hyperpolarization and arterial dilation via Ca2+-induced Ca2+ release in response to an endothelial-derived factor.     

Caveolae localize protein kinase A signaling to arterial ATP-sensitive potassium channels

Arterial ATP-sensitive K+ (K(ATP)) channels are critical regulators of vascular tone, forming a focal point for signaling by many vasoactive transmitters that alter smooth muscle contractility and so blood flow. Clinically, these channels form the target of antianginal and antihypertensive drugs, and their genetic disruption leads to hypertension and sudden cardiac death through coronary vasospasm. However, whereas the biochemical basis of K(ATP) channel modulation is well-studied, little is known about the structural or spatial organization of the signaling pathways that converge on these channels. In this study, we use discontinuous sucrose density gradients and Western blot analysis to show that K(ATP) channels localize with an upstream signaling partner, adenylyl cyclase, to smooth muscle membrane fractions containing caveolin, a protein found exclusively in cholesterol and sphingolipid-enriched membrane invaginations known as caveolae. Furthermore, we show that an antibody against the K(ATP) pore-forming subunit, Kir6.1 co-immunoprecipitates caveolin from arterial homogenates, suggesting that Kir6.1 and caveolin exist together in a complex. To assess whether the colocalization of K(ATP) channels and adenylyl cyclase to smooth muscle caveolae has functional significance, we disrupt caveolae with the cholesterol-depleting agent, methyl-beta-cyclodextrin. This reduces the cAMP-dependent protein kinase A-sensitive component of whole-cell K(ATP) current, indicating that the integrity of caveolae is important for adenylyl cyclase-mediated channel modulation. These results suggest that to be susceptible to protein kinase A-dependent activation, arterial K(ATP) channels need to be localized in the same lipid compartment as adenylyl cyclase; the results also provide the first indication of the spatial organization of signaling pathways that regulate K(ATP) channel activity.     

正常肠系膜动脉平滑肌细胞钙激活钾通道活性的观察

为了解人体正常肠系膜动脉平滑肌细胞钙激活钾通道的特性 ,取 2 4例人体正常肠系膜动脉小枝节段 ,用酶消化法获取标本细胞 ,以膜片钳制技术检测钙激活钾通道的活性 ,通过Pclamp专用软件实时采样记录其平均开放时间、平均关闭时间及平均开放概率等。结果发现 ,人体肠系膜动脉平滑肌细胞钙激活钾通道开放具有明显电压依赖性 ,在对称性高钾液中 ,电流 -电压关系曲线在 10～ 6 0mV范围内可被直线拟合。在细胞吸附式膜片和内面向外膜片中 ,通道电导分别为 192 .3± 2 9.2Ps和 2 0 2 .5± 5 8.3Ps。开放概率和开放数目随Ca2 + 浓度的增加而增加 ,膜内面应用四乙胺可减少通道开放概率及电流幅值。提示人体肠系膜动脉平滑肌细胞钙激活钾通道与人体其他血管相似 ,主要为大电导钙激活钾通道 ,具有电压和钙浓度双重依赖性。     

Spontaneous coronary vasospasm in KATP mutant mice arises from a smooth muscle-extrinsic process

In the vasculature, ATP-sensitive potassium channels (KATP) channels regulate vascular tone. Mice with targeted gene disruptions of KATP subunits expressed in vascular smooth muscle develop spontaneous coronary vascular spasm and sudden death. From these models, it was hypothesized that the loss of KATP channel activity in arterial vascular smooth muscle was responsible for coronary artery spasm. We now tested this hypothesis using a transgenic strategy where the full-length sulfonylurea receptor containing exon 40 was expressed under the control of a smooth muscle-specific SM22alpha promoter. Two transgenic founder lines were generated and independently bred to sulfonylurea receptor 2 (SUR2) null mice to generate mice that restored expression of KATP channels in vascular smooth muscle. Transgenic expression of the sulfonylurea receptor in vascular smooth muscle cells was confirmed by detecting mRNA and protein from the transgene. Functional restoration was determined by recording pinacidil-based KATP current by whole cell voltage clamping of isolated aortic vascular smooth muscle cells isolated from the transgenic restored mice. Despite successful restoration of KATP channels in vascular smooth muscle, transgene-restored SUR2 null mice continued to display frequent episodes of spontaneous ST segment elevation, identical to the phenotype seen in SUR2 null mice. As in SUR2 null mice, ST segment elevation was frequently followed by atrioventricular heart block. ST segment elevation and coronary perfusion pressure in the restored mice did not differ significantly between transgene-negative and transgene-positive SUR2 null mice. We conclude that spontaneous coronary vasospasm and sudden death in SUR2 null mice arises from a coronary artery vascular smooth muscle-extrinsic process.