Heme is a carbon monoxide receptor for large-conductance Ca2+-activated K+ channels

Carbon monoxide (CO) is an endogenous paracrine and autocrine gaseous messenger that regulates physiological functions in a wide variety of tissues. CO induces vasodilation by activating arterial smooth muscle large-conductance Ca2+-activated potassium (BK(Ca)) channels. However, the mechanism by which CO activates BK(Ca) channels remains unclear. Here, we tested the hypothesis that CO activates BK(Ca) channels by binding to channel-bound heme, a BK(Ca) channel inhibitor, and altering the interaction between heme and the conserved heme-binding domain (HBD) of the channel alpha subunit C terminus. Data obtained using thin-layer chromatography, spectrophotometry, mass spectrometry (MS), and MS-MS indicate that CO modifies the binding of reduced heme to the alpha subunit HBD. In contrast, CO does not alter the interaction between the HBD and oxidized heme (hemin), to which CO cannot bind. Consistent with these findings, electrophysiological measurements of native and cloned (cbv) cerebral artery smooth muscle BK(Ca) channels show that CO reverses BK(Ca) channel inhibition by heme but not by hemin. Site-directed mutagenesis of the cbv HBD from CKACH to CKASR abolished both heme-induced channel inhibition and CO-induced activation. Furthermore, on binding CO, heme switches from being a channel inhibitor to an activator. These findings indicate that reduced heme is a functional CO receptor for BK(Ca) channels, introduce a unique mechanism by which CO regulates the activity of a target protein, and reveal a novel process by which a gaseous messenger regulates ion channel activity.     

Endothelin blocks ATP-sensitive K+ channels and depolarizes smooth muscle cells of porcine coronary artery.

ATP-sensitive K+ channels with a conductance of 30 pS in smooth muscle cells of porcine coronary artery were found to be highly active in the intact cell-attached patch configuration when the pipette contained a physiological concentration of Ca2+ (greater than 10(-4) M). In the inside-out configuration, these channels were activated by extracellular Ca2+ and blocked by cytosolic ATP and glibenclamide. Endothelin applied to the pipette specifically blocked these channels in a concentration-dependent manner in the cell-attached configuration (half-maximal inhibition, 1.3 x 10(-9) M). A K+ channel opener, nicorandil, activated these channels even in the presence of 10(-8) M endothelin. In the whole-cell current-clamp method, the cell membrane was depolarized by endothelin and then repolarized by nicorandil. The membrane depolarization is closely related to contraction of smooth muscle cells. These results suggest that the ATP-sensitive K+ channels are important in controlling the vascular tone of the coronary artery and that endothelin can increase vascular tone by blocking these channels.     

冠状动脉平滑肌细胞大电导钙离子激活钾通道电流的特点

目的探讨正常大鼠冠状动脉平滑肌细胞大电导钙离子激活钾通道（BK通道）电流的特点,为研究疾病状况下冠状动脉平滑肌细胞BK通道电流异常变化提供正常对照。方法酶消化法分离大鼠冠状动脉平滑肌细胞;采用不同阻滞剂,对冠状动脉血管平滑肌细胞上钾通道进行鉴定;采用全细胞和单通道膜片钳实验技术分别记录冠状动脉平滑肌细胞BK通道电流,计算开放幅度和电导,观察BK通道电压敏感性和钙敏感性及加入特异性BK通道阻滞剂IBTX后BK通道电流的变化。结果正常冠状动脉平滑肌细胞BK通道电流约占总钾离子流65％±4％（t／,=12）,BK通道电导为（258±42）pS（n=6）,在刺激电位150mV时,电流密度为（275±40）pA／pF（n=8）;在电极外液钙离子浓度为1μmol／L,刺激电位为0、20、40、60、80、100、120、140和160mV条件下,BK通道开放概率（NP0）分别为0、0．0002、0．0016±0．0005、0．0283±0．0081、0．05694±0．0102、0．3533±0．0514、1．4922±0．1578、2．5975±0．3632和4．6041±0．7834（P〈0．05,n=5）;在刺激电位60mV,电极外液钙离子浓度为0、0．001、0．01、0．1、1、10、50和100μmol／L条件下,BK通道NP。分别为0、0．0001、0．0031±0．0008、0．0042±0．0090、0．0808±0．0105、0．7591±0．1274、2．7242±0．4612和3．2366±0．5728（P〈0．05,n=6）。结论BK通道广泛分布于冠状动脉平滑肌细胞上,具有电压敏感性和钙敏感性,对冠状动脉血管张力调节起重要作用。     

Cell membrane stretch activates intermediate-conductance Ca2+-activated K+ channels in arterial smooth muscle cells

The aim of this study is to determine the signal transduction of membrane stretch on intermediate-conductance Ca(2+)-activated K(+) (IKca) channels in rat aorta smooth muscle cells using the patch-clamp technique. To stretch the cell membrane, both suction to the rear end of patch pipette and hypotonic shock were used. In cell-attached and inside-out patch configurations, the open probability of IKca channels increased when 20- to 45-mmHg suction was applied. Hyposmotic swelling efficiently increased IKca channel current. When the Ca(2+)-free solution was superfused, the activation of IKca current by the hyposmotic swelling was reduced. Furthermore, gadolinium (Gd(3+)) attenuated the activation of IKca channels induced by hyposmotic swelling, whereas nicardipine did not. In the experiments with Ca(2+)-free bath solution, pretreatment with GF109203X, a protein kinase C (PKC) inhibitor, completely abolished the stretch-induced activation of IKca currents. The stretch-induced activation of IKca channels was strongly inhibited by cytochalasin D, indicating a role for the F-actin in modulation of IKca channels by changes in cell stretching. These data suggest that cell membrane stretch activates IKca channels. In addition, the activation is associated with extracellular Ca(2+) influx through stretch-activated nonselective cation channels, and is also modulated by the F-actin cytoskeleton and the activation of PKC.     

Acid-sensing ion channels interact with and inhibit BK K+ channels

Acid-sensing ion channels (ASICs) are neuronal non-voltage-gated cation channels that are activated when extracellular pH falls. They contribute to sensory function and nociception in the peripheral nervous system, and in the brain they contribute to synaptic plasticity and fear responses. Some of the physiologic consequences of disrupting ASIC genes in mice suggested that ASIC channels might modulate neuronal function by mechanisms in addition to their H(+)-evoked opening. Within ASIC channel's large extracellular domain, we identified sequence resembling that in scorpion toxins that inhibit K(+) channels. Therefore, we tested the hypothesis that ASIC channels might inhibit K(+) channel function by coexpressing ASIC1a and the high-conductance Ca(2+)- and voltage-activated K(+) (BK) channel. We found that ASIC1a associated with BK channels and inhibited their current. Reducing extracellular pH disrupted the association and relieved the inhibition. BK channels, in turn, altered the kinetics of ASIC1a current. In addition to BK, ASIC1a inhibited voltage-gated Kv1.3 channels. Other ASIC channels also inhibited BK, although acidosis-dependent relief of inhibition varied. These results reveal a mechanism of ion channel interaction and reciprocal regulation. Finding that a reduced pH activated ASIC1a and relieved BK inhibition suggests that extracellular protons may enhance the activity of channels with opposing effects on membrane voltage. The wide and varied expression patterns of ASICs, BK, and related K(+) channels suggest broad opportunities for this signaling system to alter neuronal function.     

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.     

New expression profiles of voltage-gated ion channels in arteries exposed to high blood pressure

The diameters of small arteries and arterioles are tightly regulated by the dynamic interaction between Ca(2+) and K(+) channels in the vascular smooth muscle cells. Calcium influx through voltage-gated Ca(2+) channels induces vasoconstriction, whereas the opening of K(+) channels mediates hyperpolarization, inactivation of voltage-gated Ca(2+) channels, and vasodilation. Three types of voltage-sensitive ion channels have been highly implicated in the regulation of resting vascular tone. These include the L-type Ca(2+) (Ca(L)) channels, voltage-gated K(+) (K(V)) channels, and high-conductance voltage- and Ca(2+)-sensitive K(+) (BK(Ca)) channels. Recently, abnormal expression profiles of these ion channels have been identified as part of the pathogenesis of arterial hypertension and other vasospastic diseases. An increasing number of studies suggest that high blood pressure may trigger cellular signaling cascades that dynamically alter the expression profile of arterial ion channels to further modify vascular tone. This article will briefly review the properties of Ca(L), K(V), and BK(Ca) channels, present evidence that their expression profile is altered during systemic hypertension, and suggest potential mechanisms by which the signal of elevated blood pressure may result in altered ion channel expression. A final section will discuss emerging concepts and opportunities for the development of new vasoactive drugs, which may rely on targeting disease-specific changes in ion channel expression as a mechanism to lower vascular tone during hypertensive diseases.     

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.     

Endothelium-independent effect of estrogen on Ca(2+)-activated K(+) channels in human coronary artery smooth muscle cells

OBJECTIVE: Postmenopausal estrogen replacement therapy lowers the incidence of cardiovascular disease, suggesting that estrogens support cardiovascular function. Estrogens dilate coronary arteries; however, little is known about the molecular basis of how estrogen affects the human coronary circulation. The cellular/molecular effects of estrogen action on human coronary smooth muscle were investigated in the present study. METHODS: Patch-clamp and fluorescent microscopy studies were performed on human coronary myocytes in the absence of endothelium. RESULTS: Estrogen increased whole-cell currents over a range of membrane potentials, and further studies indicated that the large-conductance (186.5 +/- 3 pS), calcium- and voltage-activated potassium (BK(Ca)) channel was the target of estrogen action. Channel activity was stimulated approximately 15-fold by nanomolar concentrations of 17 beta-estradiol, and this stimulation was reversed >90% by inhibiting cGMP-dependent protein kinase activity with 300 nM KT5823. 17 beta-Estradiol increased the level of cGMP and nitric oxide in human myocytes, and the stimulatory effect of estrogen on channel activity and NO production was reversed by inhibiting NO synthase with 10 microM N(G)-monomethyl-L-arginine. CONCLUSIONS: Our cellular and molecular studies identify the BK(Ca) channel as a target of estrogen action in human coronary artery smooth muscle. This response to estrogen involves cGMP-dependent phosphorylation of the BK(Ca) channel or a closely associated regulatory molecule, and further evidence suggests involvement of the NO/cGMP signaling system in coronary smooth muscle. These findings are the first to provide direct evidence for a molecular mechanism that can account for endothelium-independent effects of estrogen on human arteries, and may also help explain why estrogens reduce myocardial ischemia and stimulate coronary blood flow in patients with diseased coronary arteries.     

Hypersensitivity of BKCa to Ca2+ sparks underlies hyporeactivity of arterial smooth muscle in shock

Large conductance Ca(2+)-activated K(+) channels (BK(Ca)) play a critical role in blood pressure regulation by tuning the vascular smooth muscle tone, and hyposensitivity of BK(Ca) to Ca(2+) sparks resulting from its altered beta1 subunit stoichiometry underlies vasoconstriction in animal models of hypertension. Here we demonstrate hypersensitivity of BK(Ca) to Ca(2+) sparks that contributes to hypotension and blunted vasoreactivity in acute hemorrhagic shock. In arterial smooth muscle cells under voltage-clamp conditions (0 mV), the amplitude and duration, but not the frequency, of spontaneous transient outward currents of BK(Ca) origin were markedly enhanced in hemorrhagic shock, resulting in a 265% greater hyperpolarizing current. Concomitantly, subsurface Ca(2+) spark frequency was either unaltered (at 0 mV) or decreased in hyperpolarized resting cells. Examining the relationship between spark and spontaneous transient outward current amplitudes revealed a hypersensitive BK(Ca) activity to Ca(2+) spark in hemorrhagic shock, whereas the spark-spontaneous transient outward current coupling fidelity was near unity in both groups. Importantly, we found an acute upregulation of the beta1 subunit of the channel, and single-channel recording substantiated BK(Ca) hypersensitivity at micromolar Ca(2+), which promotes the alpha and beta1 subunit interaction. Treatment of shock animals with the BK(Ca) inhibitors iberiotoxin and charybdotoxin partially restored vascular membrane potential and vasoreactivity to norepinephrine and blood reinfusion. Thus, the results underscore a dynamic regulation of the BK(Ca)-Ca(2+) spark coupling and its therapeutic potential in hemorrhagic shock-associated vascular disorders.     

TRPV2 is a component of osmotically sensitive cation channels in murine aortic myocytes

Changes in membrane tension resulting from membrane stretch represent one of the key elements in blood flow regulation in vascular smooth muscle. However, the molecular mechanisms involved in the regulation of membrane stretch remain unclear. In this study, we provide evidence that a vanilloid receptor (TRPV) homologue, TRPV2 is expressed in vascular smooth muscle cells, and demonstrate that it can be activated by membrane stretch. Cell swelling caused by hypotonic solutions activated a nonselective cation channel current (NSCC) and elevated intracellular Ca2+ ([Ca2+]i) in freshly isolated cells from mouse aorta. Both of these signals were blocked by ruthenium red, an effective blocker of TRPVs. The absence of external Ca2+ abolished this increase in [Ca2+]i caused by the hypotonic stimulation and reduced the activation of NSCC. Significant immunoreactivity to mouse TRPV2 protein was detected in single mouse aortic myocytes. Moreover, the expression of TRPV2 was found in mesenteric and basilar arterial myocytes. Treatment of mouse aorta with TRPV2 antisense oligonucleotides resulted in suppression of hypotonic stimulation-induced activation of NSCC and elevation of [Ca2+]i as well as marked inhibition of TRPV2 protein expression. In Chinese hamster ovary K1 (CHO) cells transfected with TRPV2 cDNA (TRPV2-CHO), application of membrane stretch through the recording pipette and hypotonic stimulation consistently activated single NSCC. Moreover, stretch of TRPV2-CHO cells cultured on an elastic silicon membrane significantly elevated [Ca2+]i. These results provide a strong basis for our purpose that endogenous TRPV2 in mouse vascular myocytes functions as a novel and important stretch sensor in vascular smooth muscles.     

The RCK1 high-affinity Ca2+ sensor confers carbon monoxide sensitivity to Slo1 BK channels

Carbon monoxide (CO) is a lethal gas, but it is also increasingly recognized as a physiological signaling molecule capable of regulating a variety of proteins. Among them, large-conductance Ca(2+)- and voltage-gated K(+) (Slo1 BK) channels, important in vasodilation and neuronal firing, have been suggested to be directly stimulated by CO. However, the molecular mechanism of the stimulatory action of CO on the Slo1 BK channel has not been clearly elucidated. We report here that CO reliably and repeatedly activates Slo1 BK channels in excised membrane patches in the absence of Ca(2+) in a voltage-sensor-independent manner. The stimulatory action of CO on the Slo1 BK channel requires an aspartic acid and two histidine residues located in the cytoplasmic RCK1 domain, and the effect persists under the conditions known to inhibit the conventional interaction between CO and heme in other proteins. We propose that CO acts as a partial agonist for the high-affinity divalent cation sensor in the RCK1 domain of the Slo1 BK channel.     

Preserved BK channel function in vasospastic myocytes from a dog model of subarachnoid hemorrhage

Cerebral vasospasm after subarachnoid hemorrhage (SAH) is due to contraction of smooth muscle cells in the cerebral arteries. The mechanism of this contraction, however, is not well understood. Smooth muscle contraction is regulated in part by membrane potential, which is determined by K+ conductance in smooth muscle. Voltage-gated (Kv) and large-conductance, Ca2+-activated K+ (BK) channels dominate arterial smooth muscle K+ conductance. Vasospastic smooth muscle cells are depolarized relative to normal cells, but whether this is due to altered Kv or BK channel function has not been determined. This study determined if BK channels are altered during vasospasm after SAH in dogs. We first characterized BK channels in basilar-artery smooth muscle using whole-cell patch clamping and single-channel recordings. Next, we compared BK channel function between normal and vasospastic cells. There were no significant differences between normal and vasospastic cells in BK current density, kinetics, Ca2+ and voltage sensitivity, single-channel conductance or apparent Ca2+ affinity. Basilar-artery myocytes had no, small- or intermediate-conductance, Ca2+-activated K+ channels. The lack of difference in BK channels between vasospastic and control cells suggests alteration(s) in other K+ channels or other ionic conductances may underlie the membrane depolarization and vasoconstriction observed during vasospasm after SAH.     

Inflammatory modulation of calcium-activated potassium channels in canine colonic circular smooth muscle cells

BACKGROUND & AIMS: The characteristics of colonic circular smooth muscle slow waves are altered during inflammation. The aim of this study was to examine whether inflammation modulates the open-state probability of Ca2+-activated K+ (KCa) channels in these cells to contribute to these alterations. METHODS: The experiments were performed on freshly dissociated single smooth muscle cells from the canine colon using standard patch clamp methods. Inflammation was induced by mucosal exposure to ethanol and acetic acid. RESULTS: Inflammation decreased the open-state probability of large-conductance KCa (BK) channels in the cell-attached and excised inside-out configurations. The voltage sensitivity of the channels was also reduced during inflammation. Inflammation had no significant effect on the large, medium, and small conductances or the unitary current levels of channel openings. However, it decreased the maximum number of simultaneous channel openings. The channels were Ca2+-dependent and were blocked by tetraethylammonium and charybdotoxin in normal and inflamed cells. CONCLUSIONS: Inflammation decreases the open-state probability of BK channels. This may partially reverse the decrease in duration and amplitude of slow waves and depolarization of membrane potential seen in inflammation.     

Activation thresholds of K(V), BK andCl(Ca) channels in smooth muscle cells in pial precapillary arterioles

We have previously shown expression of voltage-gated K+ channels (K(V)) in smooth muscle of cerebral arterioles and suggested the channels function to oppose voltage-dependent Ca2+ entry. However, other studies indicate that large conductance Ca2+-activated K+ (BK) channels serve this function and chloride (Cl-) channels may have the opposite effect. In this study we compared the activation thresholds and absolute current amplitudes for K(V) channels, BK channels and Cl- channels at physiological membrane potentials in intact precapillary arterioles from the rabbit cerebral circulation. Patch-clamp recordings were made to measure current and membrane potential, and a video scan line was used to detect external diameter. Two strategies to determine the basal current-voltage relationship of BK channels showed the channels contributed current only at voltages positive of -35 mV, even though voltage-dependent Ca2+-entry occurred. Ca2+-activated and niflumic acid-sensitive Cl- current was detected but, between -50 and -10 mV, both BK and Cl- channel currents were much smaller and contributed less to the membrane potential compared with K(V) channel current. Furthermore, in the absence of an exogenous vasoconstrictor agent, block of K(V) channels but not BK or Cl- channels caused constriction, although in the presence of endothelin-1 block of BK or K(V) channels caused constriction. The data indicate K(V) channels are the first inhibitory mechanism to activate when there is depolarisation in precapillary arteriolar smooth muscle cells of the cerebral circulation.     

Angiotensin II activates intermediate-conductance Ca2+ -activated K+ channels in arterial smooth muscle cells

Angiostensin II (Ang II) regulates the migration and proliferation of vascular smooth muscle cells. Recent studies indicate that intermediate-conductance Ca2+ -activated K+ (IKca) channels have an important role in cell migration and proliferation. It is not known, however, whether the action of Ang II is linked to IKca channel regulation. Here, we investigated the modulation of IKca channels by Ang II in artery smooth muscle cells. Functional IKca channel expression in cultured embryonic rat aorta smooth muscle (A10) cells was studied using the patch-clamp technique. These cells predominantly express IKca channels. In contrast, large-conductance Ca2+ -activated K+ (BKca) currents were rarely observed in excised patches. Ang II increased the IKca current in a contration-dependent manner. Losartan (1.0 microM), an AT1 selective antagonist, abolished the activation of IKca channels by Ang II. Pretreatment with 100 microM myristoylated protein kinase C inhibitor peptide 20-28 or 10 microM GF109203X completely abolished the AngII-induced activation of IKca currents, whereas the action of Ang II was not prevented in the presence of 100 microM Rp-cyclic 3', 5'-hydrogen phosphotiate adenosine triethylammonium, a protein kinase A inhibitor, or 1.0 microM KT-5823, a protein kinase G inhibitor. A membrane permeant analogue of diacylglycerol 1, 2-dioctanoyl-sn-glycerol (10 microM) induced the activation of IKca currents. These data suggest that Ang II activates IKca channels through the activation of protein kinase C, and the AT1 receptor is involved in the regulation of these channels.     

PGE2 action in human coronary artery smooth muscle: role of potassium channels and signaling cross-talk

Cyclic AMP-stimulating agents are powerful vasodilators, but our knowledge of the signal transduction mechanisms of these agents, particularly in human arteries, is limited. We now report direct molecular effects of prostaglandin E(2) (PGE(2)) on cultured human coronary artery smooth muscle cells (HCASMC). Patch-clamp studies revealed that 10 microM PGE(2) opens a high-conductance (approximately 200 pS), calcium-stimulated potassium (BK(Ca)) channel in intact HCASMC. In contrast, PGE(2) had no direct effect on channels in cell-free patches, indicating involvement of a soluble second messenger. Enzyme immunoassay demonstrated that PGE(2) enhances production of cAMP in HCASMC, but does not increase [cGMP]. Furthermore, forskolin, CPT-cAMP, or CPT-cGMP mimicked the stimulatory effect of PGE(2) on BK(Ca) channel activity. Interestingly, the response to PGE(2) was unaffected by inhibiting the cAMP-dependent protein kinase, but was antagonized by inhibitors of the cGMP-dependent protein kinase (PKG). Furthermore, cAMP-stimulated PKG activity mimicked the effect of PGE(2). These studies suggest a novel PGE(2) action in human arteries: opening of BK(Ca) channels via cAMP cross-activation of PKG in HCASMC. It is proposed that this signaling mechanism may mediate the vasodilatory response to cAMP-dependent agents in the human coronary and other vascular beds.     

糖尿病对冠状动脉平滑肌细胞大电导钙激活钾通道的影响

目的 探讨糖尿病对冠状动脉平滑肌细胞大电导钙激活钾通道(BK通道)的影响变化,阐明糖尿病冠状动脉损伤的分子机制.方法 采用链脲霉素腹腔内注射建立大鼠糖尿病动物模型,酶消化法分离冠状动脉平滑肌细胞,全细胞膜片钳实验技术和Western blot分别记录和测定正常和糖尿病大鼠冠状动脉平滑肌细胞BK通道电流和亚基的表达;采用荧光测定方法测定正常和糖尿病大鼠冠状动脉平滑肌细胞内钙离子浓度.结果 当刺激电压＞100 mV时,糖尿病冠状动脉平滑肌细胞BK通道电流密度明显低于正常冠状动脉平滑肌细胞BK通道电流密度(P＜0.05),在刺激电压为150 mV时,电流密度分别为(275±40)pA/pF(n=8)和(70±10)pA/pF(n=6);与正常组比较,糖尿病组BK通道α亚基蛋白表达差异无统计学意义(P＞0.05),但β1亚基蛋白表达较低(P＜0.05);正常组和糖尿病组冠状动脉平滑肌细胞内钙离于浓度分别为(92±7)nmol/L(n=5)和(151±18)nmol/L(n=6),差异有统计学意义(P＜0.05).结论 糖尿病冠状动脉平滑肌细胞BK通道β1亚基表达下调、BK通道电流密度下降及细胞内钙离子浓度升高可能是糖尿病冠状动脉功能损伤的重要原因.     

Crosstalk between voltage-independent Ca2+ channels and L-type Ca2+ channels in A7r5 vascular smooth muscle cells at elevated intracellular pH: evidence for functional coupling between L-type Ca2+ channels and a 2-APB-sensitive cation channel

This study was designed to investigate the role of voltage-independent and voltage-dependent Ca2+ channels in the Ca2+ signaling associated with intracellular alkalinization in A7r5 vascular smooth muscle cells. Extracellular administration of ammonium chloride (20 mmol/L) resulted in elevation of intracellular pH and activation of a sustained Ca2+ entry that was inhibited by 2-amino-ethoxydiphenyl borate (2-APB, 200 micromol/L) but not by verapamil (10 micro;mol/L). Alkalosis-induced Ca2+ entry was mediated by a voltage-independent cation conductance that allowed permeation of Ca2+ (PCa/PNa approximately 6), and was associated with inhibition of L-type Ca2+ currents. Alkalosis-induced inhibition of L-type Ca2+ currents was dependent on the presence of extracellular Ca2+ and was prevented by expression of a dominant-negative mutant of calmodulin. In the absence of extracellular Ca2+, with Ba2+ or Na+ as charge carrier, intracellular alkalosis failed to inhibit but potentiated L-type Ca2+ channel currents. Inhibition of Ca2+ currents through voltage-independent cation channels by 2-APB prevented alkalosis-induced inhibition of L-type Ca2+ currents. Similarly, 2-APB prevented vasopressin-induced activation of nonselective cation channels and inhibition of L-type Ca2+ currents. We suggest the existence of a pH-controlled Ca2+ entry pathway that governs the activity of smooth muscle L-type Ca2+ channels due to control of Ca2+/calmodulin-dependent negative feedback regulation. This Ca2+ entry pathway exhibits striking similarity with the pathway activated by stimulation of phospholipase-C-coupled receptors, and may involve a similar type of cation channel. We demonstrate for the first time the tight functional coupling between these voltage-independent Ca2+ channels and classical voltage-gated L-type Ca2+ channels.     

Stimulatory and inhibitory regulation of calcium-activated potassium channels by guanine nucleotide-binding proteins.

The regulation of membrane ion channels by guanine nucleotide-binding proteins (G proteins) has been described in numerous tissues. This regulation has been shown to involve the membrane-delimited stimulatory action of G proteins on ion channels. We now show that single calcium-activated potassium channels (KCa channels) in airway smooth muscle cells are both stimulated and inhibited by G proteins in membrane patches. We demonstrate that the beta-adrenergic agonist isoproterenol stimulates channel activity via the alpha subunit of the stimulatory G protein of adenylyl cyclase, Gs, and that channel opening is inhibited by the action of the muscarinic agonist methacholine, acting via a pertussis toxin-sensitive G protein. Isoproterenol stimulated and methacholine inhibited channel activity in the same outside-out patches when GTP was present at the cytosolic surface of the patch. In inside-out patches, addition of GTP and guanosine 5'-[gamma-thio]triphosphate (GTP[gamma S]) augmented channel activity when isoproterenol was included in the patch pipette, and inhibited channel activity when methacholine was included in the pipette. Consistent with these results, in the presence of GTP[gamma S], the alpha subunit of Gs (alpha s.GTP[gamma S] complex) opened KCa channels in a dose-dependent manner, whereas in the presence of guanosine 5'-[beta-thio]diphosphate, alpha s had no effect. By contrast, application of activated alpha i or alpha o proteins did not inhibit channel activity in inside-out patches, indicating that channel inhibition is more complex than a simple alpha subunit/channel interaction, similar to the complex inhibitory regulation of adenylyl cyclase. These results suggest that hormonal regulation of KCa channels shares substantial features with the regulation of adenylyl cyclase and demonstrate that a single ion channel may serve as the regulatory target for the membrane-delimited action of stimulatory and inhibitory G proteins. Moreover, they demonstrate a potentially important functional pathway by which beta-adrenergic and other Gs-linked receptors stimulate relaxation of smooth muscle, independent of cAMP-dependent protein phosphorylation.