BKCa Currents Research Articles

Intracellular Na+ modulates large conductance Ca2+-activated K+ currents in human umbilical vein endothelial cells

We studied the effects of Na(+) influx on large-conductance Ca(2+)-activated K(+) (BK(Ca)) channels in cultured human umbilical vein endothelial cells (HUVECs) by means of patch clamp and SBFI microfluorescence measurements. In current-clamped HUVECs, extracellular Na(+) replacement by NMDG(+) or mannitol hyperpolarized cells. In voltage-clamped HUVECs, changing membrane potential from 0 mV to negative potentials increased intracellular Na(+) concentration ([Na(+)](i)) and vice versa. In addition, extracellular Na(+) depletion decreased [Na(+)](i). In voltage-clamped cells, BK(Ca) currents were markedly increased by extracellular Na(+) depletion. In inside-out patches, increasing [Na(+)](i) from 0 to 20 or 40 mM reduced single channel conductance but not open probability (NPo) of BK(Ca) channels and decreasing intracellular K(+) concentration ([K(+)](i)) gradually from 140 to 70 mM reduced both single channel conductance and NPo. Furthermore, increasing [Na(+)](i) gradually from 0 to 70 mM, by replacing K(+), markedly reduced single channel conductance and NPo. The Na(+)-Ca(2+) exchange blocker Ni(2+) or KB-R7943 decreased [Na(+)](i) and increased BK(Ca) currents simultaneously, and the Na(+) ionophore monensin completely inhibited BK(Ca) currents. BK(Ca) currents were significantly augmented by increasing extracellular K(+) concentration ([K(+)](o)) from 6 to 12 mM and significantly reduced by decreasing [K(+)](o) from 12 or 6 to 0 mM or applying the Na(+)-K(+) pump inhibitor ouabain. These results suggest that intracellular Na(+) inhibit single channel conductance of BK(Ca) channels and that intracellular K(+) increases single channel conductance and NPo.

Epiandrosterone activates BKCa channel in bovine coronary artery smooth muscle cells

Epiandrosterone (EPI), a metabolite of testosterone precursor dehydroepiandrosterone, causes relaxation of vascular smooth muscle (VSM) associated with inhibition of glucose‐6‐phosphate dehydrogenase (G6PD), which increases cellular reducing potential by converting NADP+ to NADPH. BKCa channel is a primary K+ channels in VSM. We studied whether EPI regulates BKCa channel and whether G6PD is involved in cultured bovine coronary artery smooth muscle cells (CASMC). Whole‐cell BKCa current (IBK) was recorded in the presence of 5.4 mM [K+]o/30 mM [K+]i and 0.1 or 1 μM [Ca2+]i. IBK was predominant K+ current in CASMC. IBK increased with moderate increase of depolarization, but large depolarizations caused inactivation presenting bell‐shaped I‐V relationship. The increase of [Ca2+]i caused negative shift of maximal BKCa conductance (GBKCa). EPI (>3 μM) increased GBKCa and shifted voltage‐dependence of activation further to a negative direction; GBKCa increased by ∼50% at 10 μM and by ∼100% at 100 μM. EPI caused hyperpolarization that was reversed by TEA. Suppression of G6PD by siRNA did not affect GBKCa and EPI caused similar modulation of IBK in the G6PD KO myocytes. EPI is a novel activator of BKCa channel and the activation is independent of its inhibitory action on G6PD. EPI‐induced increase in IBK may cause relaxation of CASMC via hyperpolarization.

Alteration of Currents of Large Conductance Calcium‐Activated Potassium Channels by Hyperkalemia in Porcine Arterial Smooth Muscle Cells

The wide use of hyperkelemic solutions in cardiac surgery including heart transplantation draws our attention on the effect of hyperkalemia on large conductance calcium‐activated potassium channels (BKCa). The BKCa of arterial smooth muscle cells (SMCs) is pivotal in the regulation of vascular tone. SMCs were enzymatically isolated from porcine coronary arteries. Primary cultures of the SMCs (usually 5–7 days) were used for patch‐clamp study. The effect of K+ with different concentrations (5.4, 20, 60, or 120 mM) was investigated on whole‐cell BKCa current with a holding potential of −60 mV (n=5 for each concentration). The rise of extracellular K+ in bath solutions significantly resulted in increase of BKCa currents in a K+‐concentration dependent manner in comparison with the control solution (5.4mM: 31.6±3.4pA/pF; 20mM: 73.6±11.4pA/pF; 60mM: 108.6±20.6pA/pF; 120mM: 135.2±20.5 pA/pF, p<0.05 one‐way ANOVA). Raise of extracellular K+ concentration increases BKCa activity of the porcine coronary arterial SMCs in a dose‐dependent manner. This may be an important part of the effect of hyperkalemic cardioplegia on the coronary circulation and therefore may have clinical implications in the heart preservation.Acknowledgments: Supported by Hong Kong RGC grant (CUHK4383/03M) and CUHK direct grants (4450171, 4450231, 2041305)

The Na +/Ca 2+ exchanger inhibitor KB-R7943 activates large-conductance Ca 2+-activated K + channels in endothelial and vascular smooth muscle cells

The effect of the selective inhibitor of Na +/Ca 2+ exchanger (NCX), KB-R7943, on large-conductance Ca 2+-activated K + (BK Ca) channels was examined in cultured human umbilical vein endothelial cells (HUVECs) and freshly isolated mouse aortic smooth muscle cells (MASMCs). In voltage-clamped cells, KB-R7943 reversibly activated BK Ca currents in HUVECs and MASMCs. The EC 50 of KB-R7943 for BK Ca current activation in HUVECs was determined to be 6.78 ± 0.7 μM. In inside-out and outside-out patches, KB-R7943 markedly increased BK Ca channel activity and slightly decreased single channel current amplitudes. In inside-out patches, KB-R7943 shifted the relationship between [Ca 2+] i and open probability ( P o) to the left; the [Ca 2+] i required to evoke half-maximal activation changed from 1220 ± 68 nM (in the absence of KB-R7943) to 620 ± 199 nM (in the presence of 10 μM KB-R7943). In addition, KB-R7943 shifted the relationship between membrane potential and P o to the left; the membrane potential to evoke half-maximal activation changed from 76.86 ± 1.09 mV (in the absence of KB-R7943) to 49.62 ± 2.55 mV (in the presence of 10 μM KB-R7943). In conclusion, KB-R7943 was found to act as a potent BK Ca channel activator, which increases the sensitivity of BK Ca channels to cytosolic free Ca 2+ and membrane potential, and thereby BK Ca channel activity. These results should be considered when KB-R7943 is used as NCX blocker.

Differential effects of estrogen and progesterone on potassium channels expressed in Xenopus oocytes

Hypothesis Potassium (K +) channel activation contributes in part to estrogen-mediated vasorelaxation. However, the underlying mechanism is still unclear. We hypothesize that estrogen increases K + currents via membrane-associated, non-genomic interaction and that steroid hormones have differential effects on different types of K + channels. Experimental Human large-conductance Ca 2+-activated K + channels (BK Ca) and human voltage-gated K + channels (K V1.5) were expressed in Xenopus oocytes, and K + currents elicited by voltage clamp were measured. Results Both 17β-estradiol and BSA-conjugated 17β-estradiol increased the BK Ca current in a concentration-dependent manner and this effect was abolished by tetraethylammonium ions and iberiotoxin (putative BK Ca channel blockers). 17β-estradiol-stimulated increase in the BK Ca current was unaffected by treatment with ICI 182,780 (classic estrogen receptor antagonist), tamoxifen (estrogen receptor agonist/antagonist), actinomycin D (RNA synthesis inhibitor), or cycloheximide (protein synthesis inhibitor). In contrast, progesterone reduced the BK Ca current in the absence or presence of NS 1619 (BK Ca channel activator). Progesterone also inhibited 17β-estradiol-stimulated increase in the BK Ca current. Finally, progesterone but not 17β-estradiol reduced the K V1.5 current. Conclusions The present results show that 17β-estradiol stimulates BK Ca channels without affecting K V1.5 channels. This effect is ICI 182,780-insensitive and is likely mediated via a membrane-bound binding site. Progesterone inhibits both BK Ca- and K V1.5-encoded currents. The present results suggest that inhibition of K + channels may contribute in part to its reported antagonism against 17β-estradiol-mediated vascular relaxation via BK Ca channels.

Changes in stretch-induced tone induced by intracellular acidosis in rabbit basilar artery: Effects on BK Ca channel activity

It is known that myogenic reactivity is a fundamental determinant of the relative constancy of blood flow through the cerebral artery. It is also known that acute alteration of pH significantly affects the cerebral circulation and, therefore, we investigated the effect of mechanism of action of intracellular acidosis on myogenic tone in rabbit basilar artery. Myogenic tone was developed by imposed stretch of basilar artery and intracellular acidosis induced by the bath application of 20 mmol/L sodium acetate. Sodium acetate caused a biphasic increase in myogenic tone. The initial component reached a peak quickly and then fell slowly to a lower steady-state significantly above basal tone. The sodium acetate-induced increase in myogenic tone was completely inhibited by elimination of external Ca 2+, or treatment of nifedipine, but not with gadolinium or NPPB. TEA (5 mmol/L) and iberiotoxin (100 nmol/L) inhibited the sodium acetate-induced increase in myogenic tone. In inside-out patch-clamp recordings, decreasing pH of the mock intracellular solution from 7.4 to 6.9 markedly inhibited BK Ca currents. Several inhibitors involved in Ca 2+ sensitization pathways, 10 − 6 mol/L Y-27632, 5 × 10 − 7 mol/L calphostin C and 10 − 5 mol/L PD98059 had no effect on the sodium acetate-induced increase in myogenic tone. These results suggest that intracellular acidosis increases stretch-induced myogenic tone in rabbit basilar artery. Furthermore, voltage-dependent Ca 2+ influx plays a key role in intracellular acidosis-induced increase in myogenic tone and may be mediated, at least in part, by inhibition of BK Ca.

THG113.31, a specific PGF2alpha receptor antagonist, induces human myometrial relaxation and BKCa channel activation

BackgroundPGF2alpha exerts a significant contractile effect on myometrium and is central to human labour. THG113.31, a specific non-competitive PGF2alpha receptor (FP) antagonist, exerts an inhibitory effect on myometrial contractility. The BKCa channel is ubiquitously encountered in human uterine tissue and plays a significant role in modulating myometrial cell membrane potential and excitability. The objective of this study was to investigate potential BKCa channel involvement in the response of human myometrium to THG113.31.MethodsSingle and whole-cell electrophysiological BKCa channel recordings from freshly dispersed myocytes, were investigated in the presence and absence of THG113.31. Functional studies investigated the effects of THG113.31 on isolated spontaneous myometrial contractions, in the presence and absence of the BKCa channel blocker, iberiotoxin.ResultsSingle channel recordings identified the BKCa channel as a target of THG113.31. THG113.31 significantly increased the open state probability of these channels [control 0.023+/-0.006; 10 microM THG113.31 0.087+/-0.012 (P = 0.009); and 50 microM THG113.31 0.1356+/-0.018 (P = 0.001)]. In addition, THG113.31 increased whole-cell BKCa currents over a range of membrane potentials, and this effect was reversed by 100 nanoM IbTX. Isometric tension studies demonstrated that THG113.31 exerted a significant concentration-dependent relaxant effect on human myometrial tissue and pre-incubation of strips with IbTX abolished this effect on spontaneously occurring contractions.ConclusionThese data suggests that activation of the BKCa channel may contribute, at least partially, to the uterorelaxant effect of THG113.31.

Large-conductance Ca 2+-activated K + currents blocked and impaired by homocysteine in human and rat mesenteric artery smooth muscle cells

Plenty of evidence suggests that increased blood levels of homocysteine (Hcy) are an independent risk factor for the development of vascular diseases, but the underlying mechanisms are not well understood. It is well known that the larger conductance Ca 2+-activated K + channels (BK Ca) play an essential role in vascular function, so the present study was conducted to determine direct effects of Hcy on BK Ca channel properties of smooth muscle cells. Whole-cell patch–clamp recordings were made in mesenteric artery smooth muscle cells isolated from normal rat and patients to investigate effects of 5, 50 and 500 μM Hcy on BK Ca, the main current mediating vascular responses in these cells. In human artery smooth muscle cells, maximum BK Ca density (measured at + 60 mV) was inhibited by about 24% ( n = 6, P < 0.05). In rat artery smooth muscle cells, maximum BK Ca density was decreased by approximately 27% in the presence of 50 μM Hcy ( n = 8, P < 0.05). In addition, when rat artery smooth muscle cells was treated with 50 μM Hcy for 24 h, maximum BK Ca density decreased by 58% ( n = 5, P < 0.05). These data suggest that Hcy significantly inhibited BK Ca currents in isolated human and rat artery smooth muscle cells. BK Ca reduced and impaired by elevated Hcy levels might contribute to abnormal vascular diseases.

Acetic acid opens large-conductance Ca 2+-activated K + channels in guinea pig detrusor smooth muscle cells

Acetic acid was found to have actions on urinary bladder smooth muscle in our routine ion channel screening assays. Numerous studies have examined the mechanisms of bladder irritation by acetic acid; however, the direct effect of acetic acid on ion channels in detrusor smooth muscle cells has not been evaluated. We used whole-cell patch-clamp techniques to examine the effect of acetic acid on large-conductance Ca 2+-activated K + channels (BK Ca) from guinea pig detrusor smooth muscle cells and CHO cells expressing recombinant human BK Caαβ 1 (CHO BK Caαβ 1) and human BK Caα (CHO BK Caα). Acetic acid activated BK Ca currents in a concentration-dependent (0.01% to 0.05% v/v) manner in all the cell systems studied. Acetic acid (0.05%) increased BK Ca current at + 30 mV by 2764 ± 918% ( n = 8) in guinea pig detrusor smooth muscle cells. Acetic acid (0.03%) shifted the V 1/2 of conductance–voltage curve by 64 ± 14 ( n = 5), 128 ± 14 ( n = 5), and 126 ± 12 mV ( n = 4) in CHO BK Caα, CHO BK Caαβ 1 and detrusor smooth muscle cells, respectively. This effect of acetic acid was found to be independent of pH and was also not produced by its salt form, sodium acetate. Automated patch-clamp experiments also showed similar activation of CHO BK Caαβ 1 by acetic acid. In conclusion, acetic acid directly activates BK Ca channels in detrusor smooth muscle cells. This novel study necessitates caution while interpreting the results from acetic acid bladder irritation model.

Endothelium-independent relaxation to raloxifene in porcine coronary artery

Although the vascular action of raloxifene has been studied in several vascular beds, the underlying mechanisms are still incompletely understood. The role of endothelium in raloxifene-induced vascular responses was controversial. The present study was designed to examine endothelium-independent effects of raloxifene in isolated porcine left circumflex coronary arteries. Arterial rings were suspended in organ baths and changes in isometric tension were measured. The large-conductance Ca 2+-activated K +(BK Ca) currents were recorded using a whole-cell patch-clamp technique. Treatment with raloxifene (1–10 μmol/l) reduced the contractions to 9,11-dideoxy-11α,9α-epoxy-methanoprostaglandin F 2α (U46619), serotonin (5-HT), endothelin-1 in normal Krebs solution and to CaCl 2 in a Ca 2+-free, high K +-containing solution. In endothelin-1-contracted rings, raloxifene (0.3 to 50 μmol/l) caused relaxations which were comparable in rings with and without endothelium. The raloxifene-induced relaxation was reduced by putative K + channel blockers, iberiotoxin and tetraethyl ammonium chloride (TEA +) in rings with and without endothelium, or by elevated extracellular K + ions (30 mmol/l K + and 60 mmol/l K +). 13-methyl-7-[9-(4,4,5,5,5-pentafluoropentylsulfinyl)nonyl]-7,8,9,11,12,13,14,15,16, 17-decahydro-6H-cyclopenta[a] phenanthrene-3,17-diol (ICI 182,780) did not affect raloxifene-induced relaxation. Raloxifene enhanced the outward BK Ca currents, which were sensitive to inhibition by iberiotoxin. In summary, the present study shows that raloxifene acutely relaxes porcine coronary arteries via an endothelium-independent mechanism without involving the ICI 182,780-sensitive estrogen receptors. Raloxifene mainly acts on the vascular smooth muscle cells to induce vasorelaxation by the inhibition of Ca 2+ channels and the activation of BK Ca channels. The former mechanism appears to play a more significant role.

The dietary flavonoid quercetin activates BKCa currents in coronary arteries via production of H2O2. Role in vasodilatation

Large conductance Ca(2+)-activated K(+) channels (BKCa) regulate coronary artery tone in vivo, play a key role in blood pressure regulation, and have been suggested as novel potential drug targets in hypertension. Quercetin exerts systemic and coronary vasodilator effects in vitro and reduces blood pressure in several rat models of hypertension, and its consumption is associated with a lower mortality rate from coronary heart disease in epidemiological studies. We hypothesized that quercetin might activate BKCa channel in isolated myocytes from rat coronary arteries and that this mechanism might be involved in its coronary artery relaxant effects. Membrane currents were measured using the whole-cell configuration of the patch-clamp technique. Contractile tension was recorded in rat coronary artery rings mounted in a myograph. Quercetin (>0.1 muM) increased the outward currents in the whole range of test potentials, hyperpolarized cell membranes, and increased the frequency of spontaneous transient outward currents (STOCs) carried by BKCa channels. These effects were abolished by the selective BKCa blocker iberiotoxin and by catalase. Quercetin increased dichlorofluorescein fluorescence in coronary arteries in a polyethylenglycol-catalase-sensitive manner, indicating that it increased cytosolic H(2)O(2). The membrane-permeable analogue of H(2)O(2)t-butylhydroperoxide mimicked the effects of quercetin on outward currents. The vasodilator effect of quercetin in isolated rat coronary arteries was partially inhibited by iberiotoxin. Quercetin increased BKCa currents via production of intracellular H(2)O(2). This effect is involved, at least partly, in the coronary vasodilator effects of quercetin.

Direct modulation of Ca 2+-activated K + current by H-89 in rabbit coronary arterial smooth muscle cells

The effects of H-89, a potent and selective inhibitor of protein kinase A (PKA) on Ca 2+-activated K + (BK Ca) channels in coronary arterial smooth muscle cells were examined using a patch-clamp technique. In inside-out configuration, H-89 increased the NP o of the BK Ca channel, but it reduced the dwell time of BK Ca currents. In whole-cell configuration, H-89 markedly increased BK Ca currents in a concentration-dependent manner. The EC 50 was 0.470 ± 0.0741 μM based on dwell time, 0.582 ± 0.0691 μM based on the NP o, and 0.519 ± 0.0295 μM based on the whole-cell current, respectively. H-85, which is an inactive form of H-89, increased BK Ca currents, similar to the result of H-89. The other PKA inhibitors (Rp-8-CPT-cAMPs and KT 5720) and protein phosphatase inhibitor (okadaic acid, 1 μM) had little effect on BK Ca currents and did not significantly alter the stimulatory effects of 1 μM H-89. These findings suggest that H-89 increases the BK Ca current independently of PKA.

The Role of BKCa Channels in Electrical Signal Encoding in the Mammalian Auditory Periphery

Large-conductance voltage- and Ca(2+)-activated K+ channels (BKCa) are involved in shaping spiking patterns in many neurons. Less is known about their role in mammalian inner hair cells (IHCs), mechanosensory cells with unusually large BKCa currents. These currents may be involved in shaping the receptor potential, implying crucial importance for the properties of afferent auditory signals. We addressed the function of BKCa by recording sound-induced responses of afferent auditory nerve (AN) fibers from mice with a targeted deletion of the pore-forming alpha-subunit of BKCa (BKalpha(-/-)) and comparing these with voltage responses of current-clamped IHCs. BKCa-mediated currents in IHCs were selectively abolished in BKalpha(-/-), whereas cochlear physiology was essentially normal with respect to cochlear sensitivity and frequency tuning.BKalpha(-/-) AN fibers showed deteriorated precision of spike timing, measured as an increased variance of first spike latency in response to tone bursts. This impairment could be explained by a slowed voltage response in the presynaptic IHC resulting from the reduced K+ conductance in the absence of BKCa. Maximum spike rates of AN fibers were reduced nearly twofold in BKalpha(-/-), contrasting with increased voltage responses of IHCs. In addition to presynaptic changes, which may be secondary to a modest depolarization of BKalpha(-/-) IHCs, this reduction in AN rates suggests a role of BKCa in postsynaptic AN neurons, which was supported by increased refractory periods. In summary, our results indicate an essential role of IHC BKCa channels for precise timing of high-frequency cochlear signaling as well as a function of BKCa in the primary afferent neuron.

K IR channels mediate parenchymal arteriole dilation to extracellular potassium

Astrocytes, in response to increases in neuron activity, release vasoactive substance(s) from perivascular endfeet to dilate cortical parenchymal arterioles (PAs) and drive local changes in cerebral blood flow. Recent evidence indicates astrocytic endfeet exhibit preferential expression of Ca2+-sensitive K+ (BKCa) channels, and we have measured significant BKCa currents from endfeet. However, potential PA smooth muscle targets for localized release of K+ from endfeet BKCa channels remain unclear. Elevation of [K+]o could dilate PAs by activating Na+/K+ATPases and/or inward rectifier K+ (KIR) channels to cause membrane hyperpolarization and a reduction in cytosolic Ca2+ concentration ([Ca2+]c). To investigate this, PAs were dissected from rat cerebral cortex and vascular smooth muscle cells were isolated for patch clamp studies. KIR currents were observed, as well as BKCa and KV currents. Ba2+–sensitive (100 μM) KIR current density at −100 mV was more pronounced for PA versus pial arteries (> 5 pA/pF difference at 60 mM [K+]o), with some PA cells exhibiting large KIR currents. Furthermore, we investigated the effects of [K+]o on pressurized (40 mm Hg) PA diameter, Em and [Ca2+]c. Increasing [K+]o from 3 to 8 mM hyperpolarized the smooth muscle, reduced [Ca2+]c and elicited a 38% dilation that could be blocked by exposure to 30 μM Ba2+, but not by the selective Na+/K+ATPase inhibitor dihydro-ouabain (30 μM). These data indicate KIR are responsible for [K+]o–induced dilations in PAs and suggest a key role for smooth muscle KIR channels in neurovascular coupling by mediating arteriolar dilations in response to neuron activation. Supported by NIH HL44455.

KMUP‐1 activates BKCa channels in basilar artery myocytes via cyclic nucleotide‐dependent protein kinases

This study investigated whether KMUP-1, a synthetic xanthine-based derivative, augments the delayed-rectifier potassium (K(DR))- or large-conductance Ca2+-activated potassium (BKCa) channel activity in rat basilar arteries through protein kinase-dependent and -independent mechanisms. Cerebral smooth muscle cells were enzymatically dissociated from rat basilar arteries. Conventional whole cell, perforated and inside-out patch-clamp electrophysiology was used to monitor K+- and Ca2+ channel activities. KMUP-1 (1 microM) had no effect on the K(DR) current but dramatically enhanced BKCa channel activity. This increased BKCa current activity was abolished by charybdotoxin (100 nM) and iberiotoxin (100 nM). Like KMUP-1, the membrane-permeable analogs of cGMP (8-Br-cGMP) and cAMP (8-Br-cAMP) enhanced the BKCa current. BKCa current activation by KMUP-1 was markedly inhibited by a soluble guanylate cyclase inhibitor (ODQ 10 microM), an adenylate cyclase inhibitor (SQ 22536 10 microM), competitive antagonists of cGMP and cAMP (Rp-cGMP, 100 microM and Rp-cAMP, 100 microM), and cGMP- and cAMP-dependent protein kinase inhibitors (KT5823, 300 nM and KT5720, 300 nM). Voltage-dependent L-type Ca2+ current was significantly suppressed by KMUP-1 (1 microM), and nearly abolished by a calcium channel blocker (nifedipine, 1 microM). In conclusion, KMUP-1 stimulates BKCa currents by enhancing the activity of cGMP-dependent protein kinase, and in part this is due to increasing cAMP-dependent protein kinase. Physiologically, this activation would result in the closure of voltage-dependent calcium channels and the relaxation of cerebral arteries.

Functional alterations in cerebrovascular K+ and Ca2+ channels are comparable between simulated microgravity rat and SHR

Exposure to microgravity leads to a sustained elevation in transmural pressure across the cerebral vasculature due to removal of hydrostatic pressure gradients. We hypothesized that ion channel remodeling in cerebral vascular smooth muscle cells (VSMCs) similar to that associated with hypertension may occur and play a role in upward autoregulation of cerebral vessels during microgravity. Sprague-Dawley rats were subjected to 4-wk tail suspension (Sus) to simulate the cardiovascular effect of microgravity. Large-conductance Ca(2+)-activated K(+) (BK(Ca)), voltage-gated K(+) (K(V)), and L-type voltage-dependent Ca(2+) (Ca(L)) currents of Sus and control (Con) rat cerebral VSMCs were investigated with a whole cell voltage-clamp technique. Under the same experimental conditions, K(V), BK(Ca), and Ca(L) currents of cerebral VSMCs from adult spontaneously hypertensive rats (SHR) and Wistar-Kyoto rats (WKY) were also investigated. K(V) current density decreased in Sus rats vs. Con rats [1.07 +/- 0.14 (n = 22) vs. 1.31 +/- 0.28 (n = 16) pA/pF at +20 mV (P < 0.05)] and BK(Ca) and Ca(L) current densities increased [BK(Ca): 1.70 +/- 0.37 (n = 23) vs. 0.88 +/- 0.22 (n = 19) pA/pF at +20 mV (P < 0.05); Ca(L): -2.17 +/- 0.21 (n = 35) vs. -1.31 +/- 0.10 (n = 26) pA/pF at +10 mV (P < 0.05)]. Similar changes were also observed in SHR vs. WKY cerebral VSMCs: K(V) current density decreased [1.03 +/- 0.33 (n = 9) vs. 1.62 +/- 0.64 (n = 9) pA/pF at +20 mV (P < 0.05)] and BK(Ca) and Ca(L) current densities increased [BK(Ca): 2.54 +/- 0.47 (n = 11) vs. 1.12 +/- 0.33 (n = 12) pA/pF at +20 mV (P < 0.05); Ca(L): -3.99 +/- 0.53 (n = 12) vs. -2.28 +/- 0.20 (n = 10) pA/pF at +20 mV (P < 0.05)]. These findings support our hypothesis, and their impact on space cardiovascular research is discussed.

Continuous inhalation of carbon monoxide attenuates hypoxic pulmonary hypertension development presumably through activation of BK channels

We tested the hypothesis that inhalation of a low concentration of exogenous carbon monoxide (CO) attenuates the development of hypoxic pulmonary artery hypertension by activation of large-conductance voltage and Ca(2+)-activated K(+) channels (BK(Ca)). The BK(Ca) activity was measured using whole-cell and inside-out patch clamp recordings in Wistar rat pulmonary artery (PA) myocytes. Pulmonary artery pressures were measured in vivo and membrane potentials were recorded in vitro in pressurized resistance arteries. Chronic CO inhalation slightly increases single-channel conductance of BK(Ca) channels and induces a large increase in the sensitivity of BK(Ca) channels to Ca(2+) of PA myocytes from normoxic and chronic hypoxic rats. Consequently, BK(Ca) currents are increased and play a more prominent role in controlling resting membrane potential of PA myocytes. Chronic CO inhalation also reduces hemodynamic changes induced by chronic hypoxia and attenuates hypoxic pulmonary artery hypertension. Chronic inhalation of CO attenuates hypoxic pulmonary artery hypertension development presumably through activation of BK(Ca) channels. These results highlight the potential use of CO as a novel avenue for research on the treatment of pulmonary artery hypertension (PAHT) in a similar manner to another gasotransmitter, nitric oxide.

Sodium cyanate-induced opening of calcium-activated potassium currents in hippocampal neuron-derived H19-7 cells

We investigated the chemical toxic agent sodium cyanate (NaOCN) on the large conductance calcium-activated potassium channels (BK Ca) on hippocampal neuron-derived H19-7 cells. The whole-cell and cell-attach configuration of patch-clamp technique were applied to investigate the BK Ca currents in H19-7 cells in the presence of NaOCN (0.3 mM). NaOCN activated BK Ca channels on H19-7 cells. The single-channel conductance of BK Ca channels was 138 ± 7 pS. The presence of NaOCN (0.3 mM) caused an obvious increase in open probability of BK Ca channels. NaOCN did not exert effect on the slope of the activation curve and stimulated the activity of BK Ca channels in a voltage-dependent fashion in H19-7 cells. The presence of paxilline or EGTA significantly reduced the BK Ca amplitude, in comparison with the presence of NaOCN. These findings suggest that during NaOCN exposure, the activation of BK Ca channels in neurons could be one of the ionic mechanisms underlying the decreased neuronal excitability and neurological disorders.

Inhibitory effect of protopine on K ATP channel subunits expressed in HEK-293 cells

Protopine is an isoquinoline alkaloid purified from Corydalis tubers and other families of medicinal plants. The purpose of the present study was to investigate the effects of protopine on K ATP channels and big conductance (BK Ca) channels. Protopine concentration-dependently inhibited K ATP channel currents in human embryonic kidney cells (HEK-293) which were cotransfected with Kir6.1 and sulfonylurea receptor 1 (SUR1) subunits, but not that with Kir6.1 cDNA transfection alone. At 25 μM, protopine reversibly decreased Kir6.1/SUR1 currents densities from −17.4±3 to −13.2±2.4 pA/pF at −60 mV ( n=5, P<0.05). The heterologously expressed mSlo-encoded BK Ca channel currents in HEK-293 cells were not affected by protopine (25 μM), although iberiotoxin (100 nM) significantly inhibited the expressed BK Ca currents ( n=5, P<0.05). In summary, protopine selectively inhibited K ATP channels by targeting on SUR1 subunit. This discovery may help design specific agents to selectively modulate the function of Kir6.1/SUR1 channel complex and facilitate the understanding of the structure–function relationship of specific subtype of K ATP channels.

Differential activation of potassium channels in cerebral and hindquarter arteries of rats during simulated microgravity.

The purpose of this study was to test the hypothesis that differential autoregulation of cerebral and hindquarter arteries during simulated microgravity is mediated or modulated by differential activation of K(+) channels in vascular smooth muscle cells (VSMCs) of arteries in different anatomic regions. Sprague-Dawley rats were subjected to 1- and 4-wk tail suspension to simulate the cardiovascular deconditioning effect due to short- and medium-term microgravity. K(+) channel function of VSMCs was studied by pharmacological methods and patch-clamp techniques. Large-conductance Ca(2+)-activated K(+) (BK(Ca)) and voltage-gated K(+) (K(v)) currents were determined by subtracting the current recorded after applications of 1 mM tetraethylammonium (TEA) and 1 mM TEA + 3 mM 4-aminopyridine (4-AP), respectively, from that of before. For cerebral vessels, the normalized contractility of basilar arterial rings to TEA, a BK(Ca) blocker, and 4-AP, a K(v) blocker, was significantly decreased after 1- and 4-wk simulated microgravity, respectively. VSMCs isolated from the middle cerebral artery branches of suspended rats had a more depolarized membrane potential (E(m)) and a smaller K(+) current density compared with those of control rats. Furthermore, the reduced total current density was due to smaller BK(Ca) and smaller K(v) current density in cerebral VSMCs after 1- and 4-wk tail suspension, respectively. For hindquarter vessels, VSMCs isolated from second- to sixth-order small mesenteric arteries of both 1- and 4-wk suspended rats had a more negative E(m) and larger K(+) current densities for total, BK(Ca), and K(v) currents. These results indicate that differential activation of K(+) channels occur in cerebral and hindquarter VSMCs during short- and medium-term simulated microgravity. It is further suggested that different profiles of channel remodeling might occur in VSMCs as one of the important underlying cellular mechanisms to mediate and modulate differential vascular adaptation during microgravity.