Activation Of Large-conductance Research Articles

A hypothesis: Bitter taste receptors as a therapeutic target for the clinical symptoms of SARS-CoV-2.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has paralysed the livelihood of the global population by inflicting higher mortality among the affected patients. Nearly the entire human physiological system can get disrupted by the virulence of SARS-CoV-2, which exemplifies the significance of discovering a potential drug target. Similar to angiotensin-converting enzyme 2 (ACE2), bitter taste receptors (T2Rs) unequivocally expressed on all vital human organs, particularly on nasal/oral respiratory tract, gastrointestinal organs, innate immune cells, heart, brain and urogenital cells are susceptible to SARS-CoV-2 virulence. Activation of T2Rs by bitter agonists restores vital functions to these organs via activation of large conductance, Ca2+-dependent potassium (K+) channels (BKca), and inducible nitric oxide synthase. T2R activation in the gustatory system can act as the first defence mechanism, primarily preventing or mitigating SARS-CoV-2 entry to the respiratory tract. Moreover, T2R activation is crucial for the improved vasodilation accompanied by the attenuation of systemic inflammation; hyper-innate immune responses; gastrointestinal disorders; defective neurological functions; acute kidney injury; and impotency witnessed in severe SARS-CoV-2 cases. This review discusses the potential for bitter taste receptors to act as drug targets for SARS-CoV-2 symptoms and the use of existing bitter agonists to restore T2R function.

Abstract WP543: Apelin Impairs Endothelium-Derived Hyperpolarizing Factor (EDHF)-Induced Relaxation of Cerebral Arteries by Inhibiting Activation of Large Conductance, Calcium-Activated K (BK Ca ) Channels in Vascular Smooth Muscle

Introduction: Apelin is a peptide found in fat cells, endothelial cells and CNS neurons. Apelin levels are increased in conditions linked to cardiovascular disease (e.g. obesity, diabetes). Apelin inhibits NO-induced relaxation of cerebral arteries (CA) but its effect on relaxation to EDHF, which often compensates for impaired NO responses in diseased arteries, is unknown. Methods and Results: Immunoblot analysis identified apelin receptor expression in human and rat CA. In isolated rat CA, A23187 (10 -9 – 10 -6 M) caused endothelium-dependent relaxation that was unaffected by inhibitors of eNOS (nitro-l-arginine (3 x 10 -5 M), guanylyl cyclase (ODQ (10 -5 M), cyclooxygenase (indomethacin (10 -5 M) and the H 2 O 2 -scavenger, PEG-catalase (500 U/ml) (n=6). A23187-induced relaxation was attenuated by MS-PPOH (10 -5 M), a selective inhibitor of cyp450-catalyzed synthesis of epoxyeicosatrienoic acids (EETs) (pD 2 =6.86 ± 0.3 vs 5.90 ± 0.1, without and with MS-PPOH; n=6; p<0.05), and abolished by 14,15-EE(Z)E (10 -5 M), a selective EET-receptor antagonist (n=7; p<0.05). Apelin (10 -7 M) inhibited A23187-induced relaxation (pD 2 =7.08 ± 0.2 vs 6.44 ± 0.1, without and with apelin; n=7; p<0.05) as well as relaxation to 11,12-EET, 14,15-EET and NS1619 (BK Ca channel opener), but had no effect on relaxation evoked by levcromakalim (K ATP channel opener). In whole cell patch clamp studies (10mv steps from -60mv to +80mv, 200ms duration) in CA smooth muscle cells, 11,12-EET (10 -5 M), 14,15-EET (10 -5 M), and NS1619 (10 -5 M) each caused an approximately 2-fold increase in BK Ca peak current density (at +80 mV; n=6; p<0.05), which was completely abrogated in the presence of apelin (10 -7 M). The effects of apelin on EET-induced relaxation and activation of BK Ca currents were mimicked by iberiotoxin (10 -7 M) (selective BK Ca channel blocker) and abolished by F13A (10 -7 M), an apelin receptor antagonist. Conclusion: Apelin regulates CA vascular tone by inhibiting endothelium-dependent relaxation evoked by an EDHF-like mediator (possibly an EET) through a mechanism involving activation of apelin receptors and inhibition of BK Ca channels in CA smooth muscle cells. These effects could increase the risk of cerebrovascular dysfunction under conditions where apelin levels are elevated.

Ginkgo biloba Pretreatment Attenuates Myocardial Ischemia-Reperfusion Injury via mitoBKCa.

Ginkgo biloba extracts (EGb) alleviate myocardial ischemia/reperfusion (MI/R) injury. However, the underlying mechanisms have not yet been characterized. This study aimed to investigate whether activation of large-conductance -activated channels at the inner mitochondrial membrane ( of cardiomyocytes is involved in Ginkgo biloba extract-mediated cardioprotection. Shuxuening injection (SXNI, 12.5ml/kg/d), a widely prescribed herbal medicine containing Ginkgo biloba extracts in China, or vehicle, was administered to C57BL/6 mice via tail vein injection for one week prior to surgical procedures. The mitoBKCa blocker paxilline (PAX) (1ml/kg, 115 nM) was administered via tail vein injection 30min prior to the onset of ischemia. The mice were randomly divided into the following groups: Sham, MI/R, MI/R+SXNI, and MI/R+SXNI+PAX. MI/R was induced by ligating the left anterior descending coronary artery for 30min with subsequent reperfusion for 24h. SXNI pretreatment conferred cardioprotective effects against MI/R injury as evidenced by reduced infarct size, improved cardiac function, and improved mitochondrial function. However, these effects were abrogated by co-administration with PAX. In addition, activation of mitoBKCa by Ginkgo biloba extract EGb761 reduced hypoxia/reoxygenation (H/R)-induced cardiomyocyte injury in vitro through the inhibition of mitochondrial fragmentation, restoration of the mitochondrial membrane potential, decreased generation of superoxide, and inhibition of apoptosis which is associated with alleviating mitochondrial overload. These results indicated that Ginkgo biloba extracts pretreatment protected against MI/R injury via activation of mitoBKCa.

Activation of Large Conductance, Calcium-Activated Potassium Channels by Nitric Oxide Mediates Apelin-Induced Relaxation of Isolated Rat Coronary Arteries.

Apelin increases coronary blood flow, cardiac contractility, and cardiac output. Based on these favorable hemodynamic effects, apelin and apelin-like analogs are being developed for treating heart failure and related disorders; however, the molecular mechanisms underlying apelin-induced coronary vasodilation are unknown. This study aimed to elucidate the signaling pathways by which apelin causes smooth muscle relaxation in coronary arteries. Receptors for apelin (APJ receptors) were expressed in coronary arteries, as determined by Western blot and polymerase chain reaction analyses. Immunofluorescence imaging studies identified APJ receptors on endothelial and smooth muscle cells. In isolated endothelial cells, apelin caused an increase in 4,5-diaminofluorescein fluorescence that was abolished by nitro-l-arginine (NLA) and F13A (H-Gln-Arg-Pro-Arg-Leu-Ser-His-Lys-Gly-Pro-Met-Pro-Ala-OH), an APJ receptor antagonist, consistent with increased nitric oxide (NO) production. In arterial rings, apelin caused endothelium-dependent relaxations that were abolished by NLA, F13A, and iberiotoxin. Neither oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) nor DT-2, a protein kinase G inhibitor, had any effect on apelin-induced relaxations, and apelin itself had no effect on intracellular cGMP accumulation in coronary arteries. Patch-clamp studies in isolated smooth muscle cells demonstrated that the NO donors, diethyl amine NONOate and sodium nitroprusside, caused increases in large conductance, calcium-activated potassium channel (BKCa) currents, which were inhibited by iberiotoxin but not ODQ. Thus, apelin causes endothelium-dependent relaxation of coronary arteries by stimulating endothelial APJ receptors and releasing NO, which acts in a cGMP-independent manner and increases BKCa activity in the underlying smooth muscle cells. These results provide a mechanistic basis for apelin-induced coronary vasodilation and may provide guidance for the future development of novel apelin-like therapeutic agents.

Apelin Alters Vasomotor Tone in Cerebral Arteries by Inhibiting Endothelium‐Dependent Relaxations that are Mediated by Activation of Large Conductance, Calcium‐Activated K (BKCa) Channels

Plasma levels of the adipokine, apelin, are elevated in obesity, which is a known risk factor for cardiovascular disease. Apelin has complex vasoactive effects inasmuch as the peptide may cause either vasodilation or vasoconstriction depending on the vascular bed and experimental conditions. The vasomotor effects of apelin in cerebral arteries are unknown. We previously reported that apelin inhibits BKCa channel currents in cerebral artery smooth muscle cells (PLoS One 8(12):e83051, 2013). Several endogenous vasodilators, including nitric oxide (NO) and endothelium‐derived hyperpolarizing factors(s) (EDHF), activate BKCa channels to cause relaxation of vascular smooth muscle cells. Here, we tested the hypothesis that apelin inhibits endothelium‐dependent relaxation of cerebral arteries. Rat isolated cerebral arterial rings were suspended in myographs and contracted with 5‐HT (10−7 M). Apelin (10−8 M × 10−6 M) itself had no direct vasomotor effect in cerebral arteries, with or without endothelium (n=5); however, bradykinin (10−9 M‐10−6 M) and A23187 (10−9 M × 10−5 M), a calcium ionophore, each caused endothelium‐dependent relaxation. Bradykinin‐induced relaxation was markedly inhibited by the NO synthase inhibitor, nitro‐l‐arginine (NLA; 3 × 10−5 M). The response to A23187 was unaffected by either NLA or the cyclooxygenase inhibitor, indomethacin (10−5 M), but was attenuated by MS‐PPOH (10−5 M), a selective inhibitor of cytochrome p450 catalyzed synthesis of epoxyeicosatrienoic acids (EETs) from arachidonic acid. The NO‐dependent response to bradykinin was inhibited in the presence of apelin (10−7 M) (pD2=8.02 ± 0.3 vs 7.04 ± 0.2, and Emax = 83 ± 4 vs 55 ± 9% relaxation, in the absence and presence of apelin, respectively; n=5; p<0.05), as was the NO‐independent, EDHF‐like response to A23187 (7.06 ± 0.2 vs 6.37± 0.1, in the absence and presence of apelin, respectively; n=6; p<0.05). Iberiotoxin (10−7 M), a selective BKCa channel blocker, had inhibitory effects that were similar to apelin on relaxations induced by bradykinin and A23187 (n=5). The APJ receptor antagonist, F13A (10−7 M), abolished the effects of apelin on bradykinin‐ and A23187‐induced relaxations. RT‐qPCR and immunoblot analyses confirmed the expression of APJ receptors in rat cerebral arteries (n=3–5). Immunofluorescence staining and confocal microscopy demonstrated that APJ receptors are present on smooth muscle cells in the cerebral arterial wall (n=3). Taken together, the results indicate that apelin regulates vascular tone in cerebral arteries by inhibiting relaxations evoked by endothelium‐derived NO, as well as an EDHF‐like mediator (possibly an EET). The inhibitory effect of apelin is likely mediated by activation of APJ receptors and inhibition of BKCa channels present on vascular smooth muscle cells. These inhibitory effects of apelin could be expected to increase the risk of cerebrovascular dysfunction during conditions when plasma levels of apelin are elevated.Support or Funding InformationSupported by grant HL124338 from the National Institutes of Health

Potential Sites of Action for Phloretin on BK Channels

Activation of large-conductance (BK) potassium channels by voltage and/or Ca2+ plays an important role in maintaining homeostasis. Reduced BK channel activity has pathophysiological consequences, suggesting BK channel openers may have therapeutic potential. Here, we focus on the mechanism of action of the BK opener phloretin. Previously, we reported the predominant effect of phloretin (100 μM) was to increase Po with greater efficacy at negative voltages (∼100-fold increase in 0 Ca2+), an effect consistent with actions on the gate and/or its coupling to voltage-sensors. Phloretin has effects on other Slo family members. Interestingly, phloretin inhibited unitary currents from heterologously-expressed mSlo3 channels. Thus, we used a chimeric approach between Slo1 (BK) and Slo3 to identify potential sites of action for phloretin. Domain exchange studies revealed the intracellular tail does not play a significant role in mediating phloretin's effects as an opener. However, chimeras with S6 domains from Slo3 were inhibited by phloretin. Activation was partially restored when only the distal end of S6 was exchanged, suggesting the proximal end of S6 is important in mediating phloretin's effects. Sequence alignment of this region revealed two residues of interest-G311 and G310. The former is the proposed gating hinge and is not conserved in Slo3. G310 provides added flexibility to S6 during gating of Slo1 and is conserved in Slo3. The effects of phloretin were tested on Slo1 G310A and G311A. Although phloretin increased nPo for both mutants, the effects were significantly less at G311A and similar to wild-type values for G310A, highlighting the importance of the gating hinge in mediating the effects of phloretin. These findings suggest the ability of phloretin to act as an opener at BK channels may require the structural arrangement and flexibility provided by the gating hinge.

Melatonin inhibits NO‐dependent activation of large conductance, calcium‐activated K channels (BKCa) in porcine coronary artery smooth muscle cells

We recently demonstrated that melatonin inhibits NO‐induced relaxation of porcine coronary arteries by activating MT2 melatonin‐receptors (JPET 336: 127, 2011). We tested the hypothesis that melatonin inhibits NO/cGMP‐dependent activation of BKCa channels in coronary artery smooth muscle cells. Melatonin (10−7M) inhibited sodium nitroprusside (SNP)‐induced relaxation of isolated coronary arteries but had no effect on relaxation induced by 8‐Br‐cGMP, a stable, cell permeable cGMP analog. ODQ (10−5 M) completely inhibited SNP‐induced relaxation, as well as the increase in intracellular cGMP levels in response to SNP. In patch clamp studies, SNP (10−5 M)‐induced increases in BKCa currents were abolished in the presence of ODQ (10−5 M). Melatonin (10−7M) inhibited both the voltage‐dependent and SNP‐induced increase in BKCa currents, but had no effect on BKCa currents elicited by 8‐Br‐cGMP (10−3M). The inhibitory effect of melatonin on the voltage‐dependent and SNP‐induced increase in BKCa currents was abolished by either the PDE inhibitor, zaprinast (10−5M), or the MT2‐receptor antagonist, 4P‐PDOT (10−7 M). Coronary artery relaxation in response to NO involves the cGMP‐dependent activation of BKCa channels. Melatonin impairs NO‐induced relaxation of porcine coronary arteries by inhibiting the cGMP‐dependent activation of BKCa channels in coronary smooth muscle cells. (Supported by NIH HL77204)

Acidosis Dilates Brain Parenchymal Arterioles by Conversion of Calcium Waves to Sparks to Activate BK Channels

Acidosis is a powerful vasodilator signal in the brain circulation. However, the mechanisms by which this response occurs are not well understood, particularly in the cerebral microcirculation. One important mechanism to dilate cerebral (pial) arteries is by activation of large-conductance, calcium-sensitive potassium (BK(Ca)) channels by local Ca(2+) signals (Ca(2+) sparks) through ryanodine receptors (RyRs). However, the role of this pathway in the brain microcirculation is not known. The objectives of this study were to determine the mechanism by which acidosis dilates brain parenchymal arterioles (PAs) and to elucidate the roles of RyRs and BK(Ca) channels in this response. Internal diameter and vascular smooth muscle cell Ca(2+) signals were measured in isolated pressurized murine PAs, using imaging techniques. In physiological pH (7.4), vascular smooth muscle cells exhibited primarily RyR-dependent Ca(2+) waves. Reducing external pH from 7.4 to 7.0 in both normocapnic and hypercapnic conditions decreased Ca(2+) wave activity, and dramatically increased Ca(2+) spark activity. Acidic pH caused a dilation of PAs which was inhibited by about 60% by BK(Ca) channel or RyR blockers, in a nonadditive manner. Similarly, dilator responses to acidosis were reduced by nearly 60% in arterioles from BK(Ca) channel knockout mice. Dilations induced by acidic pH were unaltered by inhibitors of K(ATP) channels or nitric oxide synthase. These results support the novel concept that acidification, by converting Ca(2+) waves to sparks, leads to the activation of BK(Ca) channels to induce dilation of cerebral PAs.

Identification of Sterol-Sensing Amino Acids in Transmembrane Domain 2 of the BK Channel Beta 1 subunit

The activity of large conductance, voltage- and calcium-gated potassium (BK) channels in myocytes critically controls arterial diameter. At <300 μM in the aqueous phase (below critical micellar concentration), lithocholate (LC) and related steroids increase myocyte BK channel activity (Po) and cause arterial dilation. We showed that: 1) BK β1 TM2 is critical for the channel sensitivity to LC, and 2) BK β2-4 cannot substitute for β1 in providing LC-sensitivity to BK channels (Bukiya et al., 2008; 2009). Moreover, computational dynamics identified two LC-sensing amino acid clusters in BK β1TM2: L157,L158,T165 and T169,L172,L173. Here, we used pinpoint mutagenesis and patch-clamp electrophysiology to determine the relative contribution of each cluster to the LC-sensitivity of BK channels. BK α (cbv1) and β1 subunits cloned from rat cerebral artery myocytes (AY330293; FJ154955) were coexpressed in Xenopus oocytes, and BK currents were recorded from inside-out patches at Ca2+i=10 μM and Vm=-20 to −40mV. BK β1T165A and β1T165A,L157A,L158A mutants rendered LC-sensitive BK channels, with 150 μM LC-induced increase in Po (×1.7 times) being identical to that found with cbv1+wtβ1. In contrast, BK β1T165A,T169A and β1T169A mutants were absolutely unresponsive to ≥150 μM LC. Data unveil a critical role for β1T169 in providing LC-sensitivity to BK channels. While the β1T169S mutation was able to rescue the channel's LC-sensitivity, the β1T169S,L172A,L173A mutation failed to do so. Moreover, BK β1L172A,L173A was also LC-insensitive. These data validate our computational prediction that hydrogen bonding between T/S169 and the steroid hydroxyl, and hydrophobic interactions between L172,L173 and the steroid rings are both necessary for successful docking of LC onto the T169,L172,L173 cluster. Thus, T169, L172 and L173 in BK β1TM2 provide a docking surface that is essential for the LC-sensitivity of BK channels.Support: HL104631 (AMD).

Antecedent Ethanol Attenuates Cerebral Ischemia/Reperfusion-induced Leukocyte-Endothelial Adhesive Interactions and Delayed Neuronal Death: Role of Large Conductance, Ca2+-activated K+Channels

EtOH-PC reduces postischemic neuronal injury in response to cerebral (I/R). We examined the mechanism underlying this protective effect by determining (i) whether it was associated with a decrease in I/R-induced leukocyte-endothelial adhesive interactions in postcapillary venules, and (ii) whether the protective effects were mediated by activation of large conductance, calcium-activated potassium (BK(Ca)) channels. Mice were administered ethanol by gavage or treated with the BK(Ca) channel opener, NS1619, 24 hours prior to I/R with or without prior treatment with the BK(Ca) channel blocker, PX. Both CCA were occluded for 20 minutes followed by two and three hours of reperfusion, and rolling (LR) and adherent (LA) leukocytes were quantified in pial venules using intravital microscopy. The extent of DND, apoptosis and glial activation in hippocampus were assessed four days after I/R. Compared with sham, I/R elicited increases in LR and LA in pial venules and DND and apoptosis as well as glial activation in the hippocampus. These effects were attenuated by EtOH-PC or antecedent NS1619 administration, and this protection was reversed by prior treatment with PX. Our results support a role for BK(Ca) channel activation in the neuroprotective effects of EtOH-PC in cerebral I/R.

Heterogeneity in function of small artery smooth muscle BKCa: involvement of the β1‐subunit

Arteriolar myogenic vasoconstriction occurs when increased stretch or membrane tension leads to smooth muscle cell depolarization and opening of voltage-gated Ca2+ channels. To prevent positive feedback and excessive pressure-induced vasoconstriction, studies in cerebral artery smooth muscle have suggested that activation of large conductance, Ca2+-activated K+ channels (BKCa) provides an opposing hyperpolarizing influence reducing Ca2+ channel activity. We have hypothesized that this mechanism may not equally apply to all vascular beds. To establish the existence of such heterogeneity in vascular reactivity, studies were performed on rat vascular smooth muscle (VSM) cells from cremaster muscle arterioles and cerebral arteries. Whole cell K+ currents were determined at pipette [Ca2+] of 100 nM or 5 microM in the presence and absence of the BKCa inhibitor, iberiotoxin (IBTX; 0.1 microM). Similar outward current densities were observed for the two cell preparations at the lower pipette Ca2+ levels. At 5 microM Ca2+, cremaster VSM showed a significantly (P < 0.05) lower current density compared to cerebral VSM (34.5 +/- 1.9 vs 45.5 +/- 1.7 pA pF(-1) at +70 mV). Studies with IBTX suggested that the differences in K+ conductance at 5 microM intracellular [Ca2+] were largely due to activity of BKCa. 17beta-Oestradiol (1 microM), reported to potentiate BKCa current via the channel's beta-subunit, caused a greater effect on whole cell K+ currents in cerebral vessel smooth muscle cells (SMCs) compared to those of cremaster muscle. In contrast, the alpha-subunit-selective BKCa opener, NS-1619 (20 microM), exerted a similar effect in both preparations. Spontaneously transient outward currents (STOCs) were more apparent (frequency and amplitude) and occurred at more negative membrane potentials in cerebral compared to cremaster SMCs. Also consistent with decreased STOC activity in cremaster SMCs was an absence of detectable Ca2+ sparks (0 of 76 cells) compared to that in cerebral SMCs (76 of 105 cells). Quantitative PCR showed decreased mRNA expression for the beta1 subunit and a decrease in the beta1:alpha ratio in cremaster arterioles compared to cerebral vessels. Similarly, cremaster arterioles showed a decrease in total BKCa protein and the beta1:alpha-subunit ratio. The data support vascular heterogeneity with respect to the activity of BKCa in terms of both beta-subunit regulation and interaction with SR-mediated Ca2+ signalling.

Cholesterol-ethanol Interactions On Vascular Myocyte BK Channels: Contribution To Alcohol-induced Cerebrovascular Constriction

The activity of large conductance, calcium- and voltage-gated potassium (BK) channels critically limits the degree of vascular smooth muscle contraction, favoring cerebrovascular dilation (Brayden and Nelson, 1992). Ethanol at levels reached in circulation after moderate binge drinking (50 mM) inhibits the activity of cerebrovascular myocyte BK channels, leading to endothelium-independent cerebrovascular constriction (Liu et al., 2004). On the other hand, high cholesterol levels, which inhibit vascular smooth muscle BK channels (Bolotina et al., 1989), decrease vascular compliance, favoring vasoconstriction (Bukiya et al., 2008). Synergistic inhibition of cerebrovascular BK channels by cholesterol and ethanol would certainly have a profound negative impact on vascular compliance and dilation. Remarkably, such synergism on channel function has not been studied. Thus, we cloned BK subunits (channel-forming cbv1 and accessory, smooth muscle-abundant β1) from rat resistance-size cerebral arteries, reconstituted the channel complex into 1-palmitoyl-2-oleoyl phosphatidylethanolamine/1-palmitoyl-2-oleoyl phosphatidylserine (POPE/POPS) bilayers, and studied cholesterol modulation of ethanol action on channel steady-state activity (NPo). Acute exposure to 50 mM ethanol mildly yet significantly decreased BK NPo (−4 ± 0.8 % from control) without modifying channel unitary conductance. In the same bilayer type, incorporation of cholesterol at levels found in cell membranes (15 % w/w) also reduced BK NPo (-8.78 ± 5.2 % from control). Remarkably, 50 mM EtOH added to the cholesterol-containing bilayer resulted in a robust decrease in BK NPo (−36 ± 8.4 % from control). These data unveil a multiplicative inhibition of BK channel activity by alcohol and cholesterol. The kinetic and biophysical mechanisms of such synergism are currently being investigated.

Role of BK‐β4 in hypo‐osmotic activation of large conductance, Ca‐sensitive K (BK) channels expressed in HEK293 cells.

BK channels, which are comprised of pore forming α and accessory β‐subunits, have been shown to eliminate intracellular K in response to hypo‐osmotic swelling. However, the role of the β subunits in this process has not been determined. We used HEK293 cells stably expressing BK‐α and examined a role for transiently expressed BK‐β4 (90% expression rate) to modulate the response of BK to cell swelling. Using patch clamp analysis, we determined NPo (cell attached, ‐Vp = −40 mV) and the % of observed channels per patch. The NPo and % channels per patch of BK‐α expressed alone in cells in isotonic (290 mOsm) solution was 0.71±0.05 (n=3) and 53%, respectively. This value was not significantly different from the NPo and % channels per patch for BK‐α in hypotonic (200 mOsm) solution (0.62±0.17, n=3 and 41%, respectively). The NPo and % channels per patch of BK‐α plus BK–β4 was significantly greater in hypotonic solution (1.32±0.15, n=3 and 76%, respectively) than in isotonic solution (0.70±0.15, n=8 and 41%, respectively). Compared to isotonic solution, when cells expressing BK‐α/β4 were in hypotonic solution, BK‐α exhibited more intense immuno‐staining at the plasma membrane and BK‐β4 was increased at the plasma membrane (western blot; OD = 0.20 in isotonic and 0.49 in hypotonic). These data suggest that the BK‐β4 subunit is necessary for trafficking of BK‐α and BK‐β4 to the plasma membrane in response to hypo‐osmotic stress.

Activation of Mitochondrial Large-Conductance Calcium-Activated K+ Channels via Protein Kinase A Mediates Desflurane-Induced Preconditioning

ATP-regulated K+ channels are involved in anesthetic-induced preconditioning (APC). The role of other K+ channels in APC is unclear. We tested the hypothesis that APC is mediated by large-conductance calcium-activated K+ channels (K(Ca)). Pentobarbital-anesthetized male C57BL/6 mice were subjected to 45 min of coronary artery occlusion and 3 h reperfusion. Thirty minutes before coronary artery occlusion, 1.0 MAC desflurane was administered for 15 min alone or in combination with the large-conductance K(Ca) channel activator NS1619 (1 microg/g i.p.), its respective vehicle dimethylsulfoxide (10 microL/g i.p.), the large-conductance K(Ca) channel blocker iberiotoxin (0.05 microg/g i.p.), or the protein kinase A (PKA) inhibitor H-89 (0.5 microg/g intraventricular). Infarct size was determined with triphenyltetrazolium chloride and area at risk with Evans blue. Mitochondrial and sarcolemmal localization of large-conductance K(Ca) channels in cardiac myocytes was investigated with immunocytochemical staining of isolated cardiac myocytes. Desflurane significantly reduced infarct size compared with control animals (7.4% +/- 0.8% vs 51.3% +/- 6.1%; P < 0.05). Activation of large-conductance K(Ca) channels by NS1619 (7.5% +/- 1.8%; P < 0.05) mimicked and blockade of large-conductance K(Ca) channels by iberiotoxin (49.1% +/- 7.5%) abrogated desflurane-induced preconditioning. PKA blockade by H-89 abolished desflurane-induced (45.1% +/- 4.0%) but not NS1619-induced (9.0% +/- 2.4%, P < 0.05) preconditioning. Immunocytochemical staining revealed that large-conductance K(Ca) channels were localized in the mitochondria but not in the sarcolemma of cardiac myocytes. These data suggest that desflurane-induced APC is mediated in part by activation of mitochondrial large-conductance K(Ca) channels, and that activation of these channels by desflurane is mediated by PKA.

Abstract 1368: BK Channel β1 Subunits Are Specific Effectors Of Cholane-induced Channel Activation

The activity of large conductance, Ca 2+ - and voltage-gated potassium (BK) channels in smooth muscle critically controls vascular tone. Depolarization-induced Ca 2+ -entry in the myocyte activates BK channels, which generate outward positive current that tends to repolarize the membrane, limit Ca 2+ entry and, thus, oppose contraction. Cholane-derived steroids (e.g., lithocholic acid, LC) reduce vascular tone in isolated, resistance-size rat cerebral arteries by selective activation of myocyte BK channels. In most tissues, native BK channels consist of pore-forming α (encoded by KCNMA1 or Slo1 ) and accessory β1–4 (encoded by KCNMB1–4 ) subunits. Remarkably, KCNMB expression is tissue-specific: while KCNMB1 is highly predominant in smooth muscle, KCNMB2–4 are not. Thus, agents that target BK β1 subunits may be used to selectively modulate myocyte BK channel function. After cloning the BK α subunit from rat cerebral artery myocytes (termed “cbv1”, AY330293 ), we demonstrated that homomeric cbv1 channel steady-state activity (NPo) was not affected by acute LC application. In contrast, heteromeric cbv1+β1 channel NPo was reversibly increased by LC (+290% of control at EC max ~150 μM; EC 50 =46 μM). Whether the other BK β subunits (2–4) can substitute for β1 to evoke LC-sensitivity in the BK channel remains unknown. To test this, we applied 150 μM LC to the intracellular side of inside-out patches excised from Xenopus laevis oocytes expressing cbv1 alone or cbv1 with a given BK β subunit subtype (1–4). Currents were evoked with the membrane clamped at ±20mV and free Ca 2+ i set to 10 μM, a concentration found in the cerebral artery myocyte during contraction. As previously found, LC consistently failed to increase homomeric cbv1 NPo, while drastically enhancing heteromeric cbv1+β1 channel NPo. Remarkably, LC failed to activate cbv1+β2, cbv1+β3 and cbv1+β4 heteromeric channels. In conclusion, the BK β1 (smooth muscle-type) subunit serves as a unique sensor for cholane-derived steroids. Thus, these compounds provide a platform for designing therapeutic agents to treat cardiovascular disease where reduction of vascular tone is required.

Functional activity of BKCa channels is not coupled to the activity of ryanodine receptors in hamster cheek pouch arterioles

The activity of large conductance, calcium-activated potassium (BKCa) channels appears to be tightly coupled to the activity of ryanodine receptors (RYR) in vascular smooth muscle (VSM) cells in myogenically active arteries. In these vessels, blockade of BKCa channels or RYR causes equivalent membrane depolarization and constriction supporting this hypothesis. However, the linkage between BKCa channels and RYR in arterioles has not been established. Hamster cheek pouch arterioles (maximum diameter = 87 μm) were isolated, cannulated with glass micropipettes, pressurized to 70 cm H2O, and internal diameter measured as an index of VSM cell contractile activity. At rest, these arterioles developed substantial myogenic tone: resting diameters at 70 cm H2O were 53 ± 4 μm largely due to Ca2+ influx through L-type calcium channels. Superfusion with 1 mM tetraethyl ammonium (TEA) to block BKCa channels, constricted the arterioles 14 ± 3 μm (n = 15, P < 0.05). In contrast, superfusion of arterioles (resting diameter = 44 ± 3 μm) with 10 μM ryanodine had no significant effect: the vessels constricted 1 ± 1 micrometer (n = 7, P > 0.05). In these same vessels, in the presence of ryanodine, 1 mM TEA constricted the arterioles 7 ± 2 μm (n = 7, P < 0.05). These data indicate that the activity of BKCa channels is not coupled to the activity of RYR receptors in hamster cheek pouch arteriolar smooth muscle cells. Based on our previous observations, we speculate that it is Ca2+ influx through L-type voltage-gated Ca2+ channels that is responsible for active BKCa channels in microvascular smooth muscle cells. Supported by PHS grant HL32469.

Activation of large-conductance, Ca2+-activated K+ channels by cannabinoids

We have examined the effects of the cannabinoid anandamide (AEA) and its stable analog, methanandamide (methAEA), on large-conductance, Ca2+-activated K+ (BK) channels using human embryonic kidney (HEK)-293 cells, in which the alpha-subunit of the BK channel (BK-alpha), both alpha- and beta1-subunits (BK-alphabeta1), or both alpha- and beta4-subunits (BK-alphabeta4) were heterologously expressed. In a whole cell voltage-clamp configuration, each cannabinoid activated BK-alphabeta1 within a similar concentration range. Because methAEA could potentiate BK-alpha, BK-alphabeta1, and BK-alphabeta4 with similar efficacy, the beta-subunits may not be involved at the site of action for cannabinoids. Under cell-attached patch-clamp conditions, application of methAEA to the bathing solution increased BK channel activity; however, methAEA did not alter channel activity in the excised inside-out patch mode even when ATP was present on the cytoplasmic side of the membrane. Application of methAEA to HEK-BK-alpha and HEK-BK-alphabeta1 did not change intracellular Ca2+ concentration. Moreover, methAEA-induced potentiation of BK channel currents was not affected by pretreatment with a CB1 antagonist (AM251), modulators of G proteins (cholera and pertussis toxins) or by application of a selective CB2 agonist (JWH133). Inhibitors of CaM, PKG, and MAPKs (W7, KT5823, and PD-98059) did not affect the potentiation. Application of methAEA to mouse aortic myocytes significantly increased BK channel currents. This study provides the first direct evidence that unknown factors in the cytoplasm mediate the ability of endogenous cannabinoids to activate BK channel currents. Cannabinoids may be hyperpolarizing factors in cells, such as arterial myocytes, in which BK channels are highly expressed.

Post-transcriptional Control of Human maxiK Potassium Channel Activity and Acute Oxygen Sensitivity by Chronic Hypoxia

Various cardiorespiratory diseases (e.g. congestive heart failure, emphysema) result in systemic hypoxia and patients consequently demonstrate adaptive cellular responses which predispose them to conditions such as pulmonary hypertension and stroke. Central to many affected excitable tissues is activity of large conductance, Ca2+-activated K+ (maxiK) channels. We have studied maxiK channel activity in HEK293 cells stably co-expressing the most widely distributed of the human alpha- and beta-subunits that constitute these channel following maneuvers which mimic severe hypoxia. At all [Ca2+]i, chronic hypoxia (approximately 18 mm Hg, 72 h) increased K+ current density, most markedly at physiological [Ca2+]i K+ currents in cells cultured in normoxia showed a [Ca2+]i-dependent sensitivity to acute hypoxic inhibition ( approximately 25 mm Hg, 3 min). However, chronic hypoxia dramatically changed the Ca2+ sensitivity of this acute hypoxic inhibitory profile such that low [Ca2+]i could sustain an acute hypoxic inhibitory response. Chronic hypoxia caused no change in alpha-subunit immunoreactivity with Western blotting but evoked a 3-fold increase in beta-subunit expression. These observations were fully supported by immunocytochemistry, which also suggested that chronic hypoxia augmented alpha/beta-subunit co-localization at the plasma membrane. Using a novel nuclear run-on assay and RNase protection we found that chronic hypoxia did not alter mRNA production rates or steady-state levels, which suggests that this important environmental cue modulates maxiK channel function via post-transcriptional mechanisms.

14,15-Epoxyeicosa-5(Z)-Enoic-mSI

Endothelium-dependent hyperpolarizations and relaxation of vascular smooth muscle induced by acetylcholine and bradykinin are mediated by endothelium-derived hyperpolarizing factors (EDHFs). In bovine coronary arteries, arachidonic acid metabolites, epoxyeicosatrienoic acids (EETs), function as EDHFs. The 14,15-EET analog 14,15-epoxyeicosa-5(Z)-enoic-methylsulfonylimide (14,15-EEZE-mSI) was synthesized and tested for agonist and antagonist activity. In U46619-preconstricted bovine coronary arterial rings, 14,15-, 11,12-, 8,9-, and 5,6-EET induced maximal concentration-related relaxation averaging 75% to 87% at 10 micromol/L, whereas, 14,15-EEZE-mSI induced maximal relaxation averaging only 7%. 14,15-EEZE-mSI (10 micromol/L) preincubation inhibited relaxation to 14,15- and 5,6- EET but not 11,12- or 8,9- EET. 14,15-EEZE-mSI also inhibited indomethacin-resistant relaxation to arachidonic acid and indomethacin-resistant and l-nitroarginine-resistant relaxation to bradykinin and methacholine. It did not alter the relaxation to sodium nitroprusside, iloprost, or the K+ channel openers bimakalim or NS1619. In cell-attached patches of isolated bovine coronary arterial smooth muscle cells, 14,15-EEZE-mSI (100 nmol/L) blocked the 14,15-EET-induced (100 nmol/L) activation of large-conductance, calcium-activated K+ channels. Mass spectrometric analysis of rat renal cortical microsomes incubated with arachidonic acid showed that 14,15-EEZE-mSI (10 micromol/L) increased EET concentrations while decreasing the concentrations of the corresponding dihydroxyeicosatrienoic acids. Therefore, 14,15-EEZE-mSI inhibits relaxation to 5,6- and 14,15- EET and the K+ channel activation by 14,15-EET. It also inhibits the EDHF component of bradykinin-induced, methacholine-induced, and arachidonic acid-induced relaxation. These results suggest that 14,15- or 5,6 -EET act as an EDHF in bovine coronary arteries.

Cholesterol antagonizes ethanol potentiation of human brain BKCa channels reconstituted into phospholipid bilayers.

The activity of large conductance, Ca2+-sensitive K+ (BKCa) channels, known to control neuronal excitability, is increased by ethanol (EtOH) exposure. Moreover, brain cholesterol (CHS) is elevated after chronic exposure to EtOH, suggesting that membrane CHS may play a role in drug tolerance. Here, we use BKCa channels from human brain (hslo subunits), reconstituted into 1-palmitoyl-2-oleoyl phosphatidylethanolamine/1-palmitoyl-2-oleoyl phosphatidylserine (POPS) bilayers, to examine CHS modulation of EtOH sensitivity. Acute exposure to clinically relevant EtOH levels increases channel activity without modifying conductance. In this minimal system, increases in CHS content within the range found in neuronal membranes lead to progressive antagonism of EtOH action. Furthermore, CHS inhibits basal channel activity with an affinity similar to that of CHS blunting of the alcohol effect. Modification of channel gating by either EtOH or CHS is reduced dramatically by removal of POPS from the bilayer, suggesting a common mechanism(s) of action. Indeed, channel dwell-time analysis indicates that CHS and EtOH exert opposite actions on the stability of channel closed states. However, each agent also acts on distinct dwell states not mirrored by the other, which contribute to the opposite effects of CHS and EtOH on channel gating.