RESUMO
Calcium-/voltage-gated, large-conductance potassium channels (BKs) control critical physiological processes, including smooth muscle contraction. Numerous observations concur that elevated membrane cholesterol (CLR) inhibits the activity of homomeric BKs consisting of channel-forming alpha subunits. In mammalian smooth muscle, however, native BKs include accessory KCNMB1 (ß1) subunits, which enable BK activation at physiological intracellular calcium. Here, we studied the effect of CLR enrichment on BK currents from rat cerebral artery myocytes. Using inside-out patches from middle cerebral artery (MCA) myocytes at [Ca2+]free=30 µM, we detected BK activation in response to in vivo and in vitro CLR enrichment of myocytes. While a significant increase in myocyte CLR was achieved within 5 min of CLR in vitro loading, this brief CLR enrichment of membrane patches decreased BK currents, indicating that BK activation by CLR requires a protracted cellular process. Indeed, blocking intracellular protein trafficking with brefeldin A (BFA) not only prevented BK activation but led to channel inhibition upon CLR enrichment. Surface protein biotinylation followed by Western blotting showed that BFA blocked the increase in plasmalemmal KCNMB1 levels achieved via CLR enrichment. Moreover, CLR enrichment of arteries with naturally high KCNMB1 levels, such as basilar and coronary arteries, failed to activate BK currents. Finally, CLR enrichment failed to activate BK channels in MCA myocytes from KCNMB1-/- mouse while activation was detected in their wild-type (C57BL/6) counterparts. In conclusion, the switch in CLR regulation of BK from inhibition to activation is determined by a trafficking-dependent increase in membrane levels of KCNMB1 subunits.
Assuntos
Subunidades beta do Canal de Potássio Ativado por Cálcio de Condutância Alta/metabolismo , Células Musculares/metabolismo , Canais de Potássio/metabolismo , Animais , Canais de Cálcio/metabolismo , Membrana Celular/metabolismo , Artérias Cerebrais/citologia , Artérias Cerebrais/metabolismo , Colesterol/metabolismo , Colesterol/fisiologia , Vasos Coronários/metabolismo , Subunidades beta do Canal de Potássio Ativado por Cálcio de Condutância Alta/fisiologia , Canais de Potássio Ativados por Cálcio de Condutância Alta/metabolismo , Masculino , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Músculo Liso Vascular/metabolismo , Canais de Potássio/fisiologia , Ratos , Ratos Sprague-Dawley , VasoconstriçãoRESUMO
Thiazide-like diuretics are the most commonly used drugs to treat arterial hypertension, with their efficacy being linked to their chronic vasodilatory effect. Previous studies suggest that activation of the large conductance voltage- and Ca2+-dependent K+ (BK) channel (Slo 1, MaxiK channel) is responsible for the thiazide-induced vasodilatory effect. But the direct electrophysiological evidence supporting this claim is lacking. BK channels can be associated with one small accessory ß-subunit (ß1-ß4) that confers specific biophysical and pharmacological characteristics to the current phenotype. The ß1-subunit is primarily expressed in smooth muscle cells (SMCs). In this study we investigated the effect of hydrochlorothiazide (HCTZ) on BK channel activity in native SMCs from human umbilical artery (HUASMCs) and HEK293T cells expressing the BK channel (with and without the ß1-subunit). Bath application of HCTZ (10 µmol/L) significantly augmented the BK current in HUASMCs when recorded using the whole-cell configurations, but it did not affect the unitary conductance and open probability of the BK channel in HUASMCs evaluated in the inside-out configuration, suggesting an indirect mechanism requiring cell integrity. In HEK293T cells expressing BK channels, HCTZ-augmented BK channel activity was only observed when the ß1-subunit was co-expressed, being concentration-dependent with an EC50 of 28.4 µmol/L, whereas membrane potential did not influence the concentration relationship. Moreover, HCTZ did not affect the BK channel current in HEK293T cells evaluated in the inside-out configuration, but significantly increases the open probability in the cell-attached configuration. Our data demonstrate that a ß1-subunit-dependent mechanism that requires SMC integrity leads to HCTZ-induced BK channel activation.
Assuntos
Hidroclorotiazida/farmacologia , Subunidades beta do Canal de Potássio Ativado por Cálcio de Condutância Alta/fisiologia , Miócitos de Músculo Liso/efeitos dos fármacos , Miócitos de Músculo Liso/fisiologia , Células Cultivadas , Humanos , Potenciais da Membrana/efeitos dos fármacosRESUMO
Large-conductance Ca(2+)- and voltage-activated K(+) (BK) channels are involved in a large variety of physiological processes. Regulatory ß-subunits are one of the mechanisms responsible for creating BK channel diversity fundamental to the adequate function of many tissues. However, little is known about the structure of its voltage sensor domain. Here, we present the external architectural details of BK channels using lanthanide-based resonance energy transfer (LRET). We used a genetically encoded lanthanide-binding tag (LBT) to bind terbium as a LRET donor and a fluorophore-labeled iberiotoxin as the LRET acceptor for measurements of distances within the BK channel structure in a living cell. By introducing LBTs in the extracellular region of the α- or ß1-subunit, we determined (i) a basic extracellular map of the BK channel, (ii) ß1-subunit-induced rearrangements of the voltage sensor in α-subunits, and (iii) the relative position of the ß1-subunit within the α/ß1-subunit complex.
Assuntos
Subunidades beta do Canal de Potássio Ativado por Cálcio de Condutância Alta/química , Animais , Transferência de Energia , Feminino , Subunidades alfa do Canal de Potássio Ativado por Cálcio de Condutância Alta/química , Subunidades alfa do Canal de Potássio Ativado por Cálcio de Condutância Alta/genética , Subunidades alfa do Canal de Potássio Ativado por Cálcio de Condutância Alta/fisiologia , Subunidades beta do Canal de Potássio Ativado por Cálcio de Condutância Alta/genética , Subunidades beta do Canal de Potássio Ativado por Cálcio de Condutância Alta/fisiologia , Modelos Moleculares , Oócitos , Conformação Proteica , Domínios Proteicos , Xenopus laevisRESUMO
The present study was designed to investigate the effect of equol on cerebral blood flow and the underlying molecular mechanisms. The regional cerebral blood flow in parietal lobe of rats was measured by using a laser Doppler flowmetry. Isolated cerebral basilar artery and mesenteric artery rings from rats were used for vascular reactivity measurement with a multi wire myography system. Outward K(+) current in smooth muscle cells of cerebral basilar artery, large-conductance Ca(2+)-activated K(+) (BK) channel current in BK-HEK 293 cells stably expressing both human α (hSlo)- and ß1-subunits, and hSlo channel current in hSlo-HEK 293 cells expressing only the α-subunit of BK channels were recorded with whole cell patch-clamp technique. The results showed that equol significantly increased regional cerebral blood flow in rats, and produced a concentration-dependent but endothelium-independent relaxation in rat cerebral basilar arteries. Both paxilline and iberiotoxin, two selective BK channel blockers, significantly inhibited equol-induced vasodilation in cerebral arteries. Outward K(+) currents in smooth muscle cells of cerebral basilar artery were increased by equol and fully reversed by washout or blockade of BK channels with iberiotoxin. Equol remarkably enhanced human BK current in BK-HEK 293 cells, but not hSlo current in hSlo-HEK 293 cells, and the increase was completely abolished by co-application of paxilline. Our findings provide the first information that equol selectively stimulates BK channel current by acting on its ß1 subunit, which may in turn contribute to the equol-mediated vasodilation and cerebral blood flow increase.
Assuntos
Circulação Cerebrovascular/efeitos dos fármacos , Equol/farmacologia , Subunidades beta do Canal de Potássio Ativado por Cálcio de Condutância Alta/fisiologia , Miócitos de Músculo Liso/efeitos dos fármacos , Vasodilatadores/farmacologia , Animais , Artérias Cerebrais/efeitos dos fármacos , Artérias Cerebrais/fisiologia , Células HEK293 , Humanos , Técnicas In Vitro , Subunidades alfa do Canal de Potássio Ativado por Cálcio de Condutância Alta/genética , Subunidades alfa do Canal de Potássio Ativado por Cálcio de Condutância Alta/fisiologia , Subunidades beta do Canal de Potássio Ativado por Cálcio de Condutância Alta/genética , Masculino , Artérias Mesentéricas/efeitos dos fármacos , Artérias Mesentéricas/fisiologia , Músculo Liso Vascular/citologia , Miócitos de Músculo Liso/fisiologia , Subunidades Proteicas/genética , Subunidades Proteicas/fisiologia , Ratos , Ratos Sprague-Dawley , Vasodilatação/efeitos dos fármacosRESUMO
Being activated by depolarizing voltages and increases in cytoplasmic Ca(2+), voltage- and calcium-activated potassium (BK) channels and their modulatory ß-subunits are able to dampen or stop excitatory stimuli in a wide range of cellular types, including both neuronal and nonneuronal tissues. Minimal alterations in BK channel function may contribute to the pathophysiology of several diseases, including hypertension, asthma, cancer, epilepsy, and diabetes. Several gating processes, allosterically coupled to each other, control BK channel activity and are potential targets for regulation by auxiliary ß-subunits that are expressed together with the α (BK)-subunit in almost every tissue type where they are found. By measuring gating currents in BK channels coexpressed with chimeras between ß1 and ß3 or ß2 auxiliary subunits, we were able to identify that the cytoplasmic regions of ß1 are responsible for the modulation of the voltage sensors. In addition, we narrowed down the structural determinants to the N terminus of ß1, which contains two lysine residues (i.e., K3 and K4), which upon substitution virtually abolished the effects of ß1 on charge movement. The mechanism by which K3 and K4 stabilize the voltage sensor is not electrostatic but specific, and the α (BK)-residues involved remain to be identified. This is the first report, to our knowledge, where the regulatory effects of the ß1-subunit have been clearly assigned to a particular segment, with two pivotal amino acids being responsible for this modulation.
Assuntos
Cálcio/metabolismo , Ativação do Canal Iônico/fisiologia , Subunidades beta do Canal de Potássio Ativado por Cálcio de Condutância Alta/fisiologia , Potássio/metabolismo , Animais , Sítios de Ligação/genética , Feminino , Humanos , Ativação do Canal Iônico/genética , Subunidades beta do Canal de Potássio Ativado por Cálcio de Condutância Alta/química , Subunidades beta do Canal de Potássio Ativado por Cálcio de Condutância Alta/genética , Lisina/química , Lisina/genética , Lisina/fisiologia , Potenciais da Membrana/genética , Potenciais da Membrana/fisiologia , Modelos Moleculares , Mutação , Oócitos/metabolismo , Oócitos/fisiologia , Estrutura Terciária de Proteína , Subunidades Proteicas/química , Subunidades Proteicas/genética , Subunidades Proteicas/fisiologia , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Xenopus laevisRESUMO
BACKGROUND AND PURPOSE: GoSlo-SR compounds are efficacious BK (KCa 1.1) channel openers, but little is known about their mechanism of action or effect on bladder contractility. We examined the effects of two closely related compounds on BK currents and bladder contractions. EXPERIMENTAL APPROACH: A combination of electrophysiology, molecular biology and synthetic chemistry was used to examine the effects of two novel channel agonists on BK channels from bladder smooth muscle cells and in HEK cells expressing BKα alone or in combination with either ß1 or ß4 subunits. KEY RESULTS: GoSlo-SR-5-6 shifted the voltage required for half maximal activation (V1/2 ) of BK channels approximately -100 mV, irrespective of the presence of regulatory ß subunits. The deaminated derivative, GoSlo-SR-5-130, also shifted the activation V1/2 in smooth muscle cells by approximately -100 mV; however, this was reduced by â¼80% in HEK cells expressing only BKα subunits. When ß1 or ß4 subunits were co-expressed with BKα, efficacy was restored. GoSlo-SR-5-130 caused a concentration-dependent reduction in spontaneous bladder contraction amplitude and this was abolished by iberiotoxin, consistent with an effect on BK channels. CONCLUSIONS AND IMPLICATIONS: GoSlo-SR-5-130 required ß1 or ß4 subunits to mediate its full effects, whereas GoSlo-SR-5-6 worked equally well in the absence or presence of ß subunits. GoSlo-SR-5-130 inhibited spontaneous bladder contractions by activating BK channels. The novel BK channel opener, GoSlo-SR-5-130, is approximately fivefold more efficacious on BK channels with regulatory ß subunits and may be a useful scaffold in the development of drugs to treat diseases such as overactive bladder.
Assuntos
Antraquinonas/farmacologia , Subunidades beta do Canal de Potássio Ativado por Cálcio de Condutância Alta/fisiologia , Subunidades Proteicas/fisiologia , Ácidos Sulfônicos/farmacologia , Animais , Células Cultivadas , Células HEK293 , Humanos , Subunidades alfa do Canal de Potássio Ativado por Cálcio de Condutância Alta/agonistas , Subunidades alfa do Canal de Potássio Ativado por Cálcio de Condutância Alta/fisiologia , Subunidades beta do Canal de Potássio Ativado por Cálcio de Condutância Alta/agonistas , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/fisiologia , Contração Muscular/efeitos dos fármacos , Contração Muscular/fisiologia , Músculo Liso/efeitos dos fármacos , Músculo Liso/fisiologia , Subunidades Proteicas/agonistas , Subunidades Proteicas/genética , Coelhos , Transfecção , Bexiga Urinária/efeitos dos fármacos , Bexiga Urinária/fisiologiaRESUMO
Our previous study demonstrated that pregnancy increased large-conductance Ca(2+)-activated potassium channel ß1 subunit (BKß1) expression and large-conductance Ca(2+)-activated potassium channel activity in uterine arteries, which were abrogated by chronic hypoxia. The present study tested the hypothesis that promoter methylation/demethylation is a key mechanism in epigenetic reprogramming of BKß1 expression patterns in uterine arteries. Ovine BKß1 promoter of 2315 bp spanning from -2211 to +104 of the transcription start site was cloned, and an Sp1-380 binding site that contains CpG dinucleotide in its core binding sequences was identified. Site-directed deletion of the Sp1 site significantly decreased the BKß1 promoter activity. Estrogen receptor-α bound to the Sp1 site through tethering to Sp1 and upregulated the expression of BKß1. The Sp1 binding site at BKß1 promoter was highly methylated in uterine arteries of nonpregnant sheep, and methylation inhibited transcription factor binding and BKß1 promoter activity. Pregnancy caused a significant decrease in CpG methylation at the Sp1 binding site and increased Sp1 binding to the BKß1 promoter and BKß1 mRNA abundance. Chronic hypoxia during gestation abrogated this pregnancy-induced demethylation and upregulation of BKß1 expression. The results provide evidence of a novel mechanism of promoter demethylation in pregnancy-induced reprogramming of large-conductance Ca(2+)-activated potassium channel expression and function in uterine arteries and suggest new insights of epigenetic mechanisms linking gestational hypoxia to aberrant uteroplacental circulation and increased risk of preeclampsia.
Assuntos
Adaptação Fisiológica/fisiologia , Subunidades beta do Canal de Potássio Ativado por Cálcio de Condutância Alta/fisiologia , Prenhez/fisiologia , Artéria Uterina/fisiologia , Animais , Ilhas de CpG/genética , Ilhas de CpG/fisiologia , Metilação de DNA , Epigênese Genética/fisiologia , Feminino , Subunidades beta do Canal de Potássio Ativado por Cálcio de Condutância Alta/genética , Modelos Animais , Gravidez , Glicoproteínas beta 1 Específicas da Gravidez/fisiologia , Ligação Proteica/fisiologia , Ovinos , Regulação para CimaRESUMO
The large conductance voltage- and Ca(2+)-activated K(+) (BK) channel is an important determinant of vascular tone and contributes to blood pressure regulation. Both activities depend on the ancillary BKß1 subunit. To determine the significance of smooth muscle BK channel activity for blood pressure regulation, we investigated the potential link between changes in arterial tone and altered blood pressure in BKß1 knockout (BKß1(-/-)) mice from three different genetically defined strains. While vascular tone was consistently increased in all BKß1(-/-) mice independent of genetic background, BKß1(-/-) strains exhibited increased (strain A), unaltered (strain B) or decreased (strain C) mean arterial blood pressures compared to their corresponding BKß1(+/+) controls. In agreement with previous data on aldosterone regulation by renal/adrenal BK channel function, BKß1(-/-) strain A mice have increased plasma aldosterone and increased blood pressure. Consistently, blockade of mineralocorticoid receptors by spironolactone treatment reversibly restored the elevated blood pressure to the BKß1(+/+) strain A level. In contrast, loss of BKß1 did not affect plasma aldosterone in strain C mice. Smooth muscle-restricted restoration of BKß1 expression increased blood pressure in BKß1(-/-) strain C mice, implying that impaired smooth muscle BK channel activity lowers blood pressure in these animals. We conclude that BK channel activity directly affects vascular tone but influences blood pressure independent of this effect via different pathways.
Assuntos
Subunidades beta do Canal de Potássio Ativado por Cálcio de Condutância Alta/fisiologia , Músculo Liso/fisiologia , Aldosterona/sangue , Animais , Aorta Torácica/fisiologia , Pressão Sanguínea/fisiologia , Técnicas In Vitro , Rim/fisiologia , Camundongos Transgênicos , Células Musculares/fisiologia , Oócitos/fisiologia , XenopusRESUMO
Vascular disease increases in incidence with age and is the commonest cause of morbidity and mortality among elderly people. Large-conductance Ca(2+)-activated K(+)(MaxiK) channel, with pore-forming α-subunit and modulatory ß1-subunit, is a key regulator of vascular tone. This study explored functional and molecular evidence of MaxiK alteration with aging in the mesenteric artery(MA). Young, Middle-aged, and Old male Wistar rats were used. Selective MaxiK channel blocker (Iberiotoxin) induced a significant increase of vascular tension in MA in all three age groups. However, these effects were greatly decreased in Old animals. The amplitude and frequency of spontaneous transient outward currents were significantly decreased with aging. Single channel recording revealed that aging induced a decrease of the open probability and the mean open time, but an increase of the mean closed time of MaxiK channel. The Ca(2+)/voltage sensitivity of MaxiK was also decreased. Western blotting showed that the protein expression of MaxiK ß1- and α-subunit was significantly reduced with aging, and the suppression of ß1 subunits was larger than that of α subunits. These data suggest that aging decreases capability of MaxiK channel in regulating vascular tone in the MA, which may be partially mediated by unparallel downregulation of α- and ß1-subunit expression.
Assuntos
Envelhecimento , Regulação para Baixo , Subunidades alfa do Canal de Potássio Ativado por Cálcio de Condutância Alta/fisiologia , Subunidades beta do Canal de Potássio Ativado por Cálcio de Condutância Alta/fisiologia , Artérias Mesentéricas/metabolismo , Animais , Pressão Sanguínea , Peso Corporal , Contração Isométrica , Masculino , Células Musculares/metabolismo , Contração Muscular , Músculo Liso Vascular/citologia , Técnicas de Patch-Clamp , Ratos , Ratos Wistar , Fatores de TempoRESUMO
Large-conductance, voltage-, and Ca²âº-dependent K⺠(BK) channels are broadly expressed in various tissues to modulate neuronal activity, smooth muscle contraction, and secretion. BK channel activation depends on the interactions among the voltage sensing domain (VSD), the cytosolic domain (CTD), and the pore gate domain (PGD) of the Slo1 α-subunit, and is further regulated by accessory ß subunits (ß1-ß4). How ß subunits fine-tune BK channel activation is critical to understand the tissue-specific functions of BK channels. Multiple sites in both Slo1 and the ß subunits have been identified to contribute to the interaction between Slo1 and the ß subunits. However, it is unclear whether and how the interdomain interactions among the VSD, CTD, and PGD are altered by the ß subunits to affect channel activation. Here we show that human ß1 and ß2 subunits alter interactions between bound Mg²âº and gating charge R213 and disrupt the disulfide bond formation at the VSD-CTD interface of mouse Slo1, indicating that the ß subunits alter the VSD-CTD interface. Reciprocally, mutations in the Slo1 that alter the VSD-CTD interaction can specifically change the effects of the ß1 subunit on the Ca²âº activation and of the ß2 subunit on the voltage activation. Together, our data suggest a novel mechanism by which the ß subunits modulated BK channel activation such that a ß subunit may interact with the VSD or the CTD and alter the VSD-CTD interface of the Slo1, which enables the ß subunit to have effects broadly on both voltage and Ca²âº-dependent activation.
Assuntos
Membrana Celular/metabolismo , Citosol/metabolismo , Ativação do Canal Iônico/fisiologia , Subunidades beta do Canal de Potássio Ativado por Cálcio de Condutância Alta/fisiologia , Animais , Membrana Celular/química , Citosol/química , Feminino , Humanos , Subunidades beta do Canal de Potássio Ativado por Cálcio de Condutância Alta/química , Camundongos , Estrutura Terciária de Proteína , Xenopus laevisRESUMO
Large-conductance voltage- and Ca(2+)-gated K(+) channels are negative-feedback regulators of excitability in many cell types. They are complexes of α subunits and of one of four types of modulatory ß subunits. These have intracellular N- and C-terminal tails and two transmembrane (TM) helices, TM1 and TM2, connected by an â¼100-residue extracellular loop. Based on endogenous disulfide formation between engineered cysteines (Cys), we found that in ß2 and ß3, as in ß1 and ß4, TM1 is closest to αS1 and αS2 and TM2 is closest to αS0. Mouse ß3 (mß3) has seven Cys in its loop, one of which is free, and this Cys readily forms disulfides with Cys substituted in the extracellular flanks of each of αS0-αS6. We identified by elimination mß3-loop Cys152 as the only free Cys. We inferred the disulfide-bonding pattern of the other six Cys. Using directed proteolysis and fragment sizing, we determined this pattern first among the four loop Cys in ß1. These are conserved in ß2-ß4, which have four additional Cys (eight in total), except that mß3 has one fewer. In ß1, disulfides form between Cys at aligned positions 1 and 8 and between Cys at aligned positions 5 and 6. In mß3, the free Cys is at position 7; position 2 lacks a Cys present in all other ß2-ß4; and the disulfide pattern is 1-8, 3-4, and 5-6. Presumably, Cys 2 cross-links to Cys 7 in all other ß2-ß4. Cross-linking of mß3 Cys152 to Cys substituted in the flanks of αS0-S5 attenuated the protection against iberiotoxin (IbTX); cross-linking of Cys152 to K296C in the αS6 flank and close to the pore enhanced protection against IbTX. In no case was N-type inactivation by the N-terminal tail of mß3 perturbed. Although the mß3 loop can move, its position with Cys152 near αK296, in which it blocks IbTX binding, is likely favored.
Assuntos
Subunidades beta do Canal de Potássio Ativado por Cálcio de Condutância Alta/análise , Subunidades beta do Canal de Potássio Ativado por Cálcio de Condutância Alta/química , Canais de Potássio Ativados por Cálcio de Condutância Alta/análise , Canais de Potássio Ativados por Cálcio de Condutância Alta/química , Subunidades Proteicas/análise , Subunidades Proteicas/química , Sequência de Aminoácidos , Animais , Cisteína/análise , Cisteína/química , Dissulfetos/análise , Dissulfetos/química , Eletrofisiologia , Células HEK293 , Humanos , Subunidades beta do Canal de Potássio Ativado por Cálcio de Condutância Alta/fisiologia , Canais de Potássio Ativados por Cálcio de Condutância Alta/fisiologia , Camundongos , Modelos Animais , Dados de Sequência Molecular , Peptídeos/farmacologia , Estrutura Terciária de Proteína/efeitos dos fármacos , Subunidades Proteicas/fisiologiaAssuntos
Canais de Potássio de Abertura Dependente da Tensão da Membrana/fisiologia , Canais de Potássio/metabolismo , Capacitação Espermática/fisiologia , Espermatozoides/fisiologia , Animais , Canais de Cálcio/fisiologia , Humanos , Subunidades alfa do Canal de Potássio Ativado por Cálcio de Condutância Alta , Subunidades beta do Canal de Potássio Ativado por Cálcio de Condutância Alta/fisiologia , Canais de Potássio Ativados por Cálcio de Condutância Alta/fisiologia , Masculino , Potenciais da Membrana/fisiologia , Proteínas de Membrana/fisiologia , Capacitação Espermática/genéticaRESUMO
The epithelial Na(+) channel (ENaC) in the aldosterone-sensitive distal nephron (ASDN) is under tonic inhibition by a local purinergic signaling system responding to changes in dietary sodium intake. Normal BK(Ca) channel function is required for flow-sensitive ATP secretion in the ASDN. We tested here whether ATP secreted through connexin channels in a coupled manner with K(+) efflux through BK(Ca) channels is required for inhibitory purinergic regulation of ENaC in response to increases in sodium intake. Inhibition of connexin channels relieves purinergic inhibition of ENaC. Deletion of the BK-ß4 regulatory subunit, which is required for normal BK(Ca) channel function and flow-sensitive ATP secretion in the ASDN, suppresses increases in urinary ATP in response to increases in sodium intake. As a consequence, ENaC activity, particularly in the presence of high sodium intake, is inappropriately elevated in BK-ß4 null mice. ENaC in BK-ß4 null mice, however, responds normally to exogenous ATP, indicating that increases in activity do not result from end-organ resistance but rather from lowered urinary ATP. Consistent with this, disruption of purinergic regulation increases ENaC activity in wild type but not BK-ß4 null mice. Consequently, sodium excretion is impaired in BK-ß4 null mice. These results demonstrate that the ATP secreted in the ASDN in a BK(Ca) channel-dependent manner is physiologically available for purinergic inhibition of ENaC in response to changes in sodium homeostasis. Impaired sodium excretion resulting form loss of normal purinergic regulation of ENaC in BK-ß4 null mice likely contributes to their elevated blood pressure.
Assuntos
Trifosfato de Adenosina/química , Canais Epiteliais de Sódio/metabolismo , Subunidades beta do Canal de Potássio Ativado por Cálcio de Condutância Alta/genética , Néfrons/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Pressão Sanguínea , Conexinas/química , Homeostase , Subunidades beta do Canal de Potássio Ativado por Cálcio de Condutância Alta/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Técnicas de Patch-Clamp , Sódio/química , Sódio/metabolismo , Sódio na Dieta/metabolismoRESUMO
BACKGROUND: Large-conductance Ca(2+) -activated K(+) (BK) channels regulate smooth muscle tone. The BK channel ß1-subunit increases Ca(2+) sensitivity of the α-subunit in smooth muscle. We studied ß1-subunit knockout (KO) mice to determine if gastrointestinal (GI) motility was altered. METHODS: Colonic and intestinal longitudinal muscle reactivity to bethanechol and colonic migrating motor complexes (CMMCs) were measured in vitro. Gastric emptying and small intestinal transit were measured in vivo. Colonic motility was assessed in vivo by measuring fecal output and glass bead expulsion time. Myoelectric activity of distal colon smooth muscle was measured in vitro using intracellular microelectrodes. KEY RESULTS: Bethanechol-induced contractions were larger in the distal colon of ß1-subunit KO compared to wild type (WT) mice; there were no differences in bethanechol reactivity in the duodenum, ileum, or proximal colon of WT vsß1-subunit KO mice. There were more retrogradely propagated CMMCs in the distal colon of ß1-subunit KO compared to WT mice. Gastrointestinal transit was unaffected by ß1-subunit KO. Fecal output was decreased and glass bead expulsion times were increased in ß1-subunit KO mice. Membrane potential of distal colon smooth muscle cells from ß1-subunit KO mice was depolarized with higher action potential frequency compared to WT mice. Paxilline (BK channel blocker) depolarized smooth muscle cells and increased action potential frequency in WT distal colon. CONCLUSIONS & INFERENCES: BK channels play a prominent role in smooth muscle function only in the distal colon of mice. Defects in smooth muscle BK channel function disrupt colonic motility causing constipation.
Assuntos
Colo/fisiologia , Motilidade Gastrointestinal/fisiologia , Subunidades beta do Canal de Potássio Ativado por Cálcio de Condutância Alta/fisiologia , Animais , Constipação Intestinal/fisiopatologia , Técnicas In Vitro , Intestino Delgado/fisiologia , Potenciais da Membrana/fisiologia , Camundongos , Camundongos Knockout , Contração Muscular/fisiologia , Músculo Liso/fisiologia , Miócitos de Músculo Liso/fisiologiaRESUMO
Our previous study demonstrated that increased Ca(2+)-activated K(+) (BK(Ca)) channel activity played a key role in the normal adaptation of reduced myogenic tone of uterine arteries in pregnancy. The present study tested the hypothesis that chronic hypoxia during gestation inhibits pregnancy-induced upregulation of BK(Ca) channel function in uterine arteries. Resistance-sized uterine arteries were isolated from nonpregnant and near-term pregnant sheep maintained at sea level (≈ 300 m) or exposed to high-altitude (3801 m) hypoxia for 110 days. Hypoxia during gestation significantly inhibited pregnancy-induced upregulation of BK(Ca) channel activity and suppressed BK(Ca) channel current density in pregnant uterine arteries. This was mediated by a selective downregulation of BK(Ca) channel ß1 subunit in the uterine arteries. In accordance, hypoxia abrogated the role of the BK(Ca) channel in regulating pressure-induced myogenic tone of uterine arteries that was significantly elevated in pregnant animals acclimatized to chronic hypoxia. In addition, hypoxia abolished the steroid hormone-mediated increase in the ß1 subunit and BK(Ca) channel current density observed in nonpregnant uterine arteries. Although the activation of protein kinase C inhibited BK(Ca) channel current density in pregnant uterine arteries of normoxic sheep, this effect was ablated in the hypoxic animals. The results demonstrate that selectively targeting BK(Ca) channel ß1 subunit plays a critical role in the maladaption of uteroplacental circulation caused by chronic hypoxia, which contributes to the increased incidence of preeclampsia and fetal intrauterine growth restriction associated with gestational hypoxia.
Assuntos
Hipóxia/fisiopatologia , Subunidades beta do Canal de Potássio Ativado por Cálcio de Condutância Alta/fisiologia , Artéria Uterina/fisiologia , Vasoconstrição/fisiologia , 4-Aminopiridina/farmacologia , Aclimatação/fisiologia , Altitude , Animais , Western Blotting , Células Cultivadas , Doença Crônica , Estradiol/farmacologia , Feminino , Técnicas In Vitro , Subunidades beta do Canal de Potássio Ativado por Cálcio de Condutância Alta/antagonistas & inibidores , Subunidades beta do Canal de Potássio Ativado por Cálcio de Condutância Alta/metabolismo , Potenciais da Membrana/efeitos dos fármacos , Células Musculares/efeitos dos fármacos , Células Musculares/metabolismo , Células Musculares/fisiologia , Peptídeos/farmacologia , Dibutirato de 12,13-Forbol/farmacologia , Bloqueadores dos Canais de Potássio/farmacologia , Gravidez , Progesterona/farmacologia , Proteína Quinase C/metabolismo , Ovinos , Tetraetilamônio/farmacologia , Fatores de Tempo , Regulação para Cima , Artéria Uterina/citologia , Artéria Uterina/metabolismo , Vasoconstrição/efeitos dos fármacosRESUMO
We determined the possible role of large-conductance Ca2+-activated K (BK) channels in regulation of venous tone in small capacitance veins and blood pressure. In rat mesenteric venous smooth muscle cells (MV SMC), BK channel α- and ß1-subunits were coexpressed, unitary BK currents were detected, and single-channel currents were sensitive to voltage and [Ca2+]i. Rat MV SMCs displayed Ca sparks and iberiotoxin-sensitive spontaneous transient outward currents. Under resting conditions in vitro, rat MV exhibited nifedipine-sensitive spontaneous oscillatory constrictions. Blockade of BK channels by paxilline and Ca2+ sparks by ryanodine constricted rat MV. Nifedipine caused venodilation and blocked paxilline-induced, KCl-induced (20 mM), and BayK8644-induced contraction. Acute inhibition of BK channels with iberiotoxin in vivo increased blood pressure and reduced venous capacitance, measured as an increase in mean circulatory filling pressure in conscious rats. BK channel α-subunits and L-type Ca2+ channel α1-C subunits are expressed in murine MV. However, these channels are not functional because murine MV lack nifedipine-sensitive basal tone and rhythmic constrictions. Murine MV were also insensitive to paxilline, ryanodine, KCl, and BayK8644, consistent with our previous studies showing that murine MV do not have BK ß1-subunits. These data show that not only there are species-dependent properties in ion channel control of venomotor tone but also BK channels are required for rhythmic oscillations in venous tone.
Assuntos
Subunidades alfa do Canal de Potássio Ativado por Cálcio de Condutância Alta/fisiologia , Subunidades beta do Canal de Potássio Ativado por Cálcio de Condutância Alta/fisiologia , Veias Mesentéricas/metabolismo , Contração Muscular/fisiologia , Músculo Liso Vascular/metabolismo , Vasodilatação/fisiologia , Animais , Sinalização do Cálcio/efeitos dos fármacos , Sinalização do Cálcio/fisiologia , Células Cultivadas , Subunidades alfa do Canal de Potássio Ativado por Cálcio de Condutância Alta/antagonistas & inibidores , Subunidades alfa do Canal de Potássio Ativado por Cálcio de Condutância Alta/biossíntese , Subunidades beta do Canal de Potássio Ativado por Cálcio de Condutância Alta/antagonistas & inibidores , Subunidades beta do Canal de Potássio Ativado por Cálcio de Condutância Alta/biossíntese , Masculino , Veias Mesentéricas/efeitos dos fármacos , Veias Mesentéricas/fisiopatologia , Camundongos , Camundongos Endogâmicos C57BL , Contração Muscular/efeitos dos fármacos , Músculo Liso Vascular/efeitos dos fármacos , Músculo Liso Vascular/fisiopatologia , Miócitos de Músculo Liso/efeitos dos fármacos , Miócitos de Músculo Liso/metabolismo , Ratos , Ratos Sprague-Dawley , Especificidade da Espécie , Vasodilatação/efeitos dos fármacosRESUMO
The large conductance calcium- and voltage-activated potassium channel (BK channel) and its smooth muscle-specific ß1 subunit regulate excitationcontraction coupling in many types of smooth muscle cells. However, the relative contribution of BK channels to control of M2- or M3-muscarinic acetylcholine receptor mediated airway smooth muscle contraction is poorly understood. Previously, we showed that knockout of the BK channel ß1 subunit enhances cholinergic-evoked trachea contractions. Here, we demonstrate that the enhanced contraction of the BK ß1 knockout can be ascribed to a defect in BK channel opposition of M2 receptor-mediated contractions. Indeed, the enhanced contraction of ß1 knockout is eliminated by specific M2 receptor antagonism. The role of BK ß1 to oppose M2 signalling is evidenced by a greater than fourfold increase in the contribution of L-type voltage-dependent calcium channels to contraction that otherwise does not occur with M2 antagonist or with ß1 containing BK channels. The mechanism through which BK channels oppose M2-mediated recruitment of calcium channels is through a negative shift in resting voltage that offsets, rather than directly opposes, M2-mediated depolarization. The negative shift in resting voltage is reduced to similar extents by BK ß1 knockout or by paxilline block of BK channels. Normalization of ß1 knockout baseline voltage with low external potassium eliminated the enhanced M2-receptor mediated contraction. In summary, these findings indicate that an important function of BK/ß1 channels is to oppose cholinergic M2 receptor-mediated depolarization and activation of calcium channels by restricting excitationcontraction coupling to more negative voltage ranges.
Assuntos
Subunidades beta do Canal de Potássio Ativado por Cálcio de Condutância Alta/fisiologia , Contração Muscular/fisiologia , Receptor Muscarínico M2/fisiologia , Traqueia/fisiologia , Animais , Canais de Cálcio Tipo L/fisiologia , Acoplamento Excitação-Contração/efeitos dos fármacos , Acoplamento Excitação-Contração/fisiologia , Técnicas In Vitro , Indóis/farmacologia , Subunidades beta do Canal de Potássio Ativado por Cálcio de Condutância Alta/antagonistas & inibidores , Subunidades beta do Canal de Potássio Ativado por Cálcio de Condutância Alta/deficiência , Subunidades beta do Canal de Potássio Ativado por Cálcio de Condutância Alta/genética , Potenciais da Membrana , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Modelos Biológicos , Antagonistas Muscarínicos/farmacologia , Contração Muscular/efeitos dos fármacos , Músculo Liso/efeitos dos fármacos , Músculo Liso/fisiologia , Pirenzepina/análogos & derivados , Pirenzepina/farmacologia , Bloqueadores dos Canais de Potássio/farmacologia , Receptor Muscarínico M2/antagonistas & inibidores , Mecânica Respiratória , Transdução de Sinais , Traqueia/efeitos dos fármacosRESUMO
Chronic hypoxia protects the heart against injury caused by acute oxygen deprivation, but its salutary mechanism is poorly understood. The aim was to find out whether cardiomyocytes isolated from chronically hypoxic hearts retain the improved resistance to injury and whether the mitochondrial large-conductance Ca2+-activated K+ (BKCa) channels contribute to the protective effect. Adult male rats were adapted to continuous normobaric hypoxia (inspired O2 fraction 0.10) for 3 wk or kept at room air (normoxic controls). Myocytes, isolated separately from the left ventricle (LVM), septum (SEPM), and right ventricle, were exposed to 25-min metabolic inhibition with sodium cyanide, followed by 30-min reenergization (MI/R). Some LVM were treated with either 30 µM NS-1619 (BKCa opener), or 2 µM paxilline (BKCa blocker), starting 25 min before metabolic inhibition. Cell injury was detected by Trypan blue exclusion and lactate dehydrogenase (LDH) release. Chronic hypoxia doubled the number of rod-shaped LVM and SEPM surviving the MI/R insult and reduced LDH release. While NS-1619 protected cells from normoxic rats, it had no additive salutary effect in the hypoxic group. Paxilline attenuated the improved resistance of cells from hypoxic animals without affecting normoxic controls; it also abolished the protective effect of NS-1619 on LDH release in the normoxic group. While chronic hypoxia did not affect protein abundance of the BKCa channel regulatory ß1-subunit, it markedly decreased its glycosylation level. It is concluded that ventricular myocytes isolated from chronically hypoxic rats retain the improved resistance against injury caused by MI/R. Activation of the mitochondrial BKCa channel likely contributes to this protective effect.
Assuntos
Hipóxia/fisiopatologia , Canais de Potássio Ativados por Cálcio de Condutância Alta/fisiologia , Mitocôndrias Cardíacas/fisiologia , Miócitos Cardíacos/fisiologia , Animais , Benzimidazóis/farmacologia , Western Blotting , Separação Celular , Sobrevivência Celular , Células Cultivadas , Doença Crônica , Glicosilação , Indóis/farmacologia , Precondicionamento Isquêmico Miocárdico , L-Lactato Desidrogenase/metabolismo , Subunidades beta do Canal de Potássio Ativado por Cálcio de Condutância Alta/fisiologia , Canais de Potássio Ativados por Cálcio de Condutância Alta/antagonistas & inibidores , Masculino , Mitocôndrias Cardíacas/efeitos dos fármacos , Traumatismo por Reperfusão Miocárdica/fisiopatologia , Traumatismo por Reperfusão Miocárdica/prevenção & controle , Miócitos Cardíacos/efeitos dos fármacos , Bloqueadores dos Canais de Potássio/farmacologia , Ratos , Ratos WistarRESUMO
Large-conductance Ca2+-activated K+ (BK) channels are composed of pore-forming α-subunits and accessory ß1-subunits that modulate Ca2+ sensitivity. BK channels regulate arterial myogenic tone and renal Na+ clearance/K+ reabsorption. Previous studies using indirect or short-term blood pressure measurements found that BK channel ß1-subunit knockout (BK ß1-KO) mice were hypertensive. We evaluated 24-h mean arterial pressure (MAP) and heart rate in BK ß1-KO mice using radiotelemetry. BK ß1-KO mice did not have a higher 24-h average MAP when compared with wild-type (WT) mice, although MAP was â¼10 mmHg higher at night. The dose-dependent peak declines in MAP by nifedipine were only slightly larger in BK ß1-KO mice. In BK ß1-KO mice, giving 1% NaCl to mice to drink for 7 days caused a transient (5 days) elevation of MAP (â¼5 mmHg); MAP returned to pre-saline levels by day 6. BK ß1-KO mesenteric arteries in vitro demonstrated diminished contractile responses to paxilline, increased reactivity to Bay K 8644 and norepinephrine (NE), and maintained relaxation to isoproterenol. Paxilline and Bay K 8644 did not constrict WT or BK ß1-KO mesenteric veins (MV). BK ß1-subunits are not expressed in MV. The results indicate that BK ß1-KO mice are not hypertensive on normal or high-salt intake. BK channel deficiency increases arterial reactivity to NE and L-type Ca2+ channel function in vitro, but the L-type Ca2+ channel modulation of MAP is not altered in BK ß1-KO mice. BK and L-type Ca(2+) channels do not modulate murine venous tone. It appears that selective loss of BK channel function in arteries only is not sufficient to cause sustained hypertension.
Assuntos
Hipertensão/genética , Subunidades beta do Canal de Potássio Ativado por Cálcio de Condutância Alta/genética , Subunidades beta do Canal de Potássio Ativado por Cálcio de Condutância Alta/fisiologia , Animais , Pressão Sanguínea/efeitos dos fármacos , Pressão Sanguínea/genética , Pressão Sanguínea/fisiologia , Peso Corporal/fisiologia , Bloqueadores dos Canais de Cálcio/farmacologia , Canais de Cálcio Tipo L/efeitos dos fármacos , Canais de Cálcio Tipo L/genética , Canais de Cálcio Tipo L/fisiologia , Coração/anatomia & histologia , Frequência Cardíaca/efeitos dos fármacos , Frequência Cardíaca/genética , Frequência Cardíaca/fisiologia , Hipertensão/fisiopatologia , Óperon Lac/genética , Subunidades beta do Canal de Potássio Ativado por Cálcio de Condutância Alta/efeitos dos fármacos , Artérias Mesentéricas/metabolismo , Artérias Mesentéricas/fisiologia , Veias Mesentéricas/efeitos dos fármacos , Veias Mesentéricas/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Contração Muscular/genética , Contração Muscular/fisiologia , Relaxamento Muscular/genética , Relaxamento Muscular/fisiologia , Músculo Liso Vascular/efeitos dos fármacos , Músculo Liso Vascular/fisiologia , Nifedipino/farmacologia , Tamanho do Órgão/fisiologia , Bloqueadores dos Canais de Potássio/farmacologia , Vasodilatação/genética , Vasodilatação/fisiologiaRESUMO
Large-conductance, Ca(2+)- and voltage-sensitive K(+) (BK) channels regulate neuronal functions such as spike frequency adaptation and transmitter release. BK channels are composed of four Slo1 subunits, which contain the voltage-sensing and pore-gate domains in the membrane and Ca(2+) binding sites in the cytoplasmic domain, and accessory ß subunits. Four types of BK channel ß subunits (ß1-ß4) show differential tissue distribution and unique functional modulation, resulting in diverse phenotypes of BK channels. Previous studies show that both the ß1 and ß2 subunits increase Ca(2+) sensitivity, but different mechanisms may underline these modulations. However, the structural domains in Slo1 that are critical for Ca(2+)-dependent activation and targeted by these ß subunits are not known. Here, we report that the N termini of both the transmembrane (including S0) and cytoplasmic domains of Slo1 are critical for ß2 modulation based on the study of differential effects of the ß2 subunit on two orthologs, mouse Slo1 and Drosophila Slo1. The N terminus of the cytoplasmic domain of Slo1, including the AC region (ßA-αC) of the RCK1 (regulator of K(+) conductance) domain and the peptide linking it to S6, both of which have been shown previously to mediate the coupling between Ca(2+) binding and channel opening, is specifically required for the ß2 but not for the ß1 modulation. These results suggest that the ß2 subunit modulates the coupling between Ca(2+) binding and channel opening, and, although sharing structural homology, the BK channel ß subunits interact with structural domains in the Slo1 subunit differently to enhance channel activity.