RESUMO
P2X1 receptors are adenosine triphosphate (ATP)-gated cation channels that are functionally important for male fertility, bladder contraction, and platelet aggregation. The activity of P2X1 receptors is modulated by lipids and intracellular messengers such as cAMP, which can stimulate protein kinase A (PKA). Exchange protein activated by cAMP (EPAC) is another cAMP effector; however, its effect on P2X1 receptors has not yet been determined. Here, we demonstrate that P2X1 currents, recorded from human embryonic kidney (HEK) cells transiently transfected with P2X1 cDNA, were inhibited by the highly selective EPAC activator 007-AM. In contrast, EPAC activation enhanced P2X2 current amplitude. The PKA activator 6-MB-cAMP did not affect P2X1 currents, but inhibited P2X2 currents. The inhibitory effects of EPAC on P2X1 were prevented by triple mutation of residues 21 to 23 on the amino terminus of P2X1 subunits to the equivalent amino acids on P2X2 receptors. Double mutation of residues 21 and 22 and single mutation of residue 23 also protected P2X1 receptors from inhibition by EPAC activation. Finally, the inhibitory effects of EPAC on P2X1 were also prevented by NSC23766, an inhibitor of Rac1, a member of the Rho family of small GTPases. These data suggest that EPAC is an important regulator of P2X1 and P2X2 receptors.
Assuntos
Proteínas Quinases Dependentes de AMP Cíclico/farmacologia , AMP Cíclico/metabolismo , Regulação da Expressão Gênica , Fatores de Troca do Nucleotídeo Guanina/farmacologia , Rim/metabolismo , Receptores Purinérgicos P2X1/metabolismo , Receptores Purinérgicos P2X2/metabolismo , Trifosfato de Adenosina , Aminoquinolinas/farmacologia , Fatores de Troca do Nucleotídeo Guanina/antagonistas & inibidores , Células HEK293 , Humanos , Rim/efeitos dos fármacos , Pirimidinas/farmacologia , Receptores Purinérgicos P2X1/genética , Receptores Purinérgicos P2X2/genética , Proteínas rac1 de Ligação ao GTP/antagonistas & inibidoresRESUMO
LINGO1 is a transmembrane protein that is up-regulated in the cerebellum of patients with Parkinson's disease (PD) and Essential Tremor (ET). Patients with additional copies of the LINGO1 gene also present with tremor. Pharmacological or genetic ablation of large conductance Ca2+-activated K+ (BK) channels also result in tremor and motor disorders. We hypothesized that LINGO1 is a regulatory BK channel subunit. We show that 1) LINGO1 coimmunoprecipitated with BK channels in human brain, 2) coexpression of LINGO1 and BK channels resulted in rapidly inactivating BK currents, and 3) LINGO1 reduced the membrane surface expression of BK channels. These results suggest that LINGO1 is a regulator of BK channels, which causes a "functional knockdown" of these currents and may contribute to the tremor associated with increased LINGO1 levels.
Assuntos
Canais de Potássio Ativados por Cálcio de Condutância Alta/metabolismo , Proteínas de Membrana/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Sequência de Aminoácidos , Linhagem Celular , Cerebelo/metabolismo , Humanos , Canais de Potássio Ativados por Cálcio de Condutância Alta/genética , Proteínas de Membrana/genética , Proteínas do Tecido Nervoso/genética , Ligação ProteicaRESUMO
Airway smooth muscle expresses abundant BKCa channels, but their role in regulating contractions remains controversial. This study examines the effects of two potent BKCa channel openers on agonist-induced phasic contractions in rabbit and mouse bronchi. First, we demonstrated the ability of 10 µM GoSlo-SR5-130 to activate BKCa channels in inside-out patches from rabbit bronchial myocytes, where it shifted the activation V1/2 by -88 ± 11 mV (100 nM Ca2+, n = 7). In mouse airway smooth muscle cells, GoSlo-SR5-130 dose dependently shifted V1/2 by 12-83 mV over a concentration range of 1-30 µM. Compound X, a racemic mixture of two enantiomers, reported to be potent BKCa channel openers, shifted V1/2 by 20-79 mV over a concentration range of 0.3-3 µM. In rabbit bronchial rings, exposure to histamine (1 µM) induced phasic contractions after a delay of ~35 min. These were abolished by GoSlo-SR5-130 (30 µM). Nifedipine (100 nM) and CaCCinhA01 (10 µM), a TMEM16A blocker, also abolished histamine-induced phasic contractions. In mouse bronchi, similar phasic contractions were evoked by exposure to U46619 (100 nM) and carbachol (100 nM). In each case, these were inhibited by concentrations of GoSlo-SR5-130 and compound X that shifted the activation V1/2 of BKCa channels in the order of -80 mV. In conclusion, membrane potential-dependent regulation of L-type Ca2+ channels appears to be important for histamine-, U46619-, and carbachol-induced phasic contractions in airway smooth muscle. Contractions can be abolished by BKCa channel openers, suggesting that these channels are potential targets for treating some causes of airway obstruction.
Assuntos
Subunidades alfa do Canal de Potássio Ativado por Cálcio de Condutância Alta/genética , Potenciais da Membrana/efeitos dos fármacos , Miócitos de Músculo Liso/metabolismo , Animais , Antraquinonas/farmacologia , Brônquios/efeitos dos fármacos , Brônquios/crescimento & desenvolvimento , Brônquios/metabolismo , Relação Dose-Resposta a Droga , Subunidades alfa do Canal de Potássio Ativado por Cálcio de Condutância Alta/agonistas , Potenciais da Membrana/genética , Camundongos , Contração Muscular/efeitos dos fármacos , Miócitos de Músculo Liso/efeitos dos fármacos , Técnicas de Patch-Clamp , Coelhos , Ácidos Sulfônicos/farmacologiaRESUMO
GoSlo-SR-5-6 is a novel large-conductance Ca(2+)-activated K(+) (BK) channel agonist that shifts the activation V1/2 of these channels in excess of -100 mV when applied at a concentration of 10 µM. Although the structure-activity relationship of this family of molecules has been established, little is known about how they open BK channels. To help address this, we used a combination of electrophysiology, mutagenesis, and mathematical modeling to investigate the molecular mechanisms underlying the effect of GoSlo-SR-5-6. Our data demonstrate that the effects of this agonist are practically abolished when three point mutations are made: L227A in the S4/S5 linker in combination with S317R and I326A in the S6C region. Our data suggest that GoSlo-SR-5-6 interacts with the transmembrane domain of the channel to enhance pore opening. The Horrigan-Aldrich model suggests that GoSlo-SR-5-6 works by stabilizing the open conformation of the channel and the activated state of the voltage sensors, yet decouples the voltage sensors from the pore gate.
Assuntos
Canais de Potássio Ativados por Cálcio de Condutância Alta/agonistas , Canais de Potássio Ativados por Cálcio de Condutância Alta/genética , Canais de Potássio Ativados por Cálcio de Condutância Alta/fisiologia , MutagêneseRESUMO
Rabbit corpus cavernosum smooth muscle (RCCSM) cells express ion channels that produce Ca2+-activated Cl- (IClCa) current, but low sensitivity to conventional antagonists has made its role in tone generation difficult to evaluate. We have reexamined this question using two new generation IClCa blockers, T16Ainh-A01 and CaCCinh-A01. Isolated RCCSM cells were studied using the perforated patch method. Current-voltage protocols revealed that both L-type Ca2+ current and IClCa T16Ainh-A01 and CaCCinh-A01 (10 µM) reduced IClCa by ~85%, while 30 µM abolished it. L-type Ca2+ current was unaffected by 10 µM CaCCinh-A01 but was reduced by 50% at 30 µM CaCCinh-A01, 46% at 10 µM T16Ainh-A01, and 78% at 30 µM T16Ainh-A01. Both drugs reduced spontaneous isometric tension in RCCSM strips, by 60-70% at 10 µM and >90% at 30 µM. Phenylephrine (PE)-enhanced tension was also reduced (ED50 = 3 µM, CaCCinh-A01; 14 µM, T16Ainh-A01). CaCCinh-A01 at 10 µM had little effect on 60 mM KCl contractures, though they were reduced by 30 µM CaCCinh-A01 and T16Ainh-A01 (10 µM and 30 µM) consistent with their effects on L-type Ca2+ current. Both drugs also reversed the stimulatory effect of PE on intracellular Ca2+ waves, studied with laser scanning confocal microscopy in isolated RCCSM cells. In conclusion, although both drugs were effective blockers of IClCa, the effect of T16Ainh-A01 on L-type Ca2+ current precludes its use for evaluating the role of IClCa in tone generation. However, 10 µM CaCCinh-A01 selectively blocked IClCa versus L-type Ca2+ current and reduced spontaneous and PE-induced tone, suggesting that IClCa is important in maintaining penile detumescence.
Assuntos
Canais de Cálcio Tipo L/fisiologia , Músculo Liso/fisiologia , Pênis/fisiologia , Animais , Bloqueadores dos Canais de Cálcio/farmacologia , Masculino , Músculo Liso/efeitos dos fármacos , Técnicas de Cultura de Órgãos , Pênis/efeitos dos fármacos , CoelhosRESUMO
Interstitial cells of Cajal (ICC) isolated from the rabbit urethra exhibit Ca2+-activated Cl- currents (I ClCa) that are important for the development of urethral tone. Here, we examined if TMEM16A (ANO1) contributed to this activity by examining the effect of "new-generation" TMEM16A inhibitors, CACCinh-A01 and T16Ainh-A01, on I ClCa recorded from freshly isolated rabbit urethral ICC (RUICC) and on contractions of intact strips of rabbit urethra smooth muscle. Real-time quantitative PCR experiments demonstrated that TMEM16A was highly expressed in rabbit urethra smooth muscle, in comparison to TMEM16B and TMEM16F. Single-cell RT-PCR experiments revealed that only TMEM16A was expressed in freshly isolated RUICC. Depolarization-evoked I ClCa in isolated RUICC, recorded using voltage clamp, were inhibited by CACCinh-A01 and T16Ainh-A01 with IC50 values of 1.2 and 3.4 µM, respectively. Similarly, spontaneous transient inward currents (STICs) recorded from RUICC voltage clamped at -60 mV and spontaneous transient depolarizations (STDs), recorded in current clamp, were also inhibited by CACCinh-A01 and T16Ainh-A01. In contrast, spontaneous Ca2+ waves in isolated RUICC were only partially reduced by CACCinh-A01 and T16Ainh-A01. Finally, neurogenic contractions of strips of rabbit urethra smooth muscle (RUSM), evoked by electric field stimulation (EFS), were also significantly reduced by CACCinh-A01 and T16Ainh-A01. These data are consistent with the idea that TMEM16A is involved with CACCs in RUICC and in contraction of rabbit urethral smooth muscle.
Assuntos
Anoctamina-1/antagonistas & inibidores , Cálcio/metabolismo , Cloretos/metabolismo , Células Intersticiais de Cajal/efeitos dos fármacos , Potenciais da Membrana/efeitos dos fármacos , Uretra/efeitos dos fármacos , Animais , Células Cultivadas , Canais de Cloreto/metabolismo , Feminino , Células Intersticiais de Cajal/metabolismo , Masculino , Músculo Liso/efeitos dos fármacos , Músculo Liso/metabolismo , Coelhos , Uretra/metabolismoRESUMO
KEY POINTS: Tonic contractions of rabbit urethra are associated with spontaneous electrical slow waves that are thought to originate in pacemaker cells termed interstitial cells of Cajal (ICC). ICC pacemaker activity results from their ability to generate propagating Ca(2+) waves, although the exact mechanisms of propagation are not understood. In this study, we have identified spontaneous localised Ca(2+) events for the first time in urethral ICC; these were due to Ca(2+) release from the endoplasmic reticulum (ER) via ryanodine receptors (RyRs) and, while they often remained localised, they sometimes initiated propagating Ca(2+) waves. We show that propagation of Ca(2+) waves in urethral ICC is critically dependent upon Ca(2+) influx via reverse mode NCX. Our data provide a clearer understanding of the intracellular mechanisms involved in the generation of ICC pacemaker activity. Interstitial cells of Cajal (ICC) are putative pacemaker cells in the rabbit urethra. Pacemaker activity in ICC results from spontaneous propagating Ca(2+) waves that are modulated by [Ca(2+)]o and whose propagation is inhibited by inositol tri-phosphate receptor (IP3 R) blockers. The purpose of this study was to further examine the role of Ca(2+) influx and Ca(2+) release in the propagation of Ca(2+) waves. Intracellular Ca(2+) was measured in Fluo-4-loaded ICC using a Nipkow spinning disc confocal microscope at fast acquisition rates (50 fps). We identified previously undetected localised Ca(2+) events originating from ryanodine receptors (RyRs). Inhibiting Ca(2+) influx by removing [Ca(2+)]o or blocking reverse mode sodium-calcium exchange (NCX) with KB-R 7943 or SEA-0400 abolished Ca(2+) waves, while localised Ca(2+) events persisted. Stimulating RyRs with 1 mm caffeine restored propagation. Propagation was also inhibited when Ca(2+) release sites were uncoupled by buffering intracellular Ca(2+) with EGTA-AM. This was reversed when Ca(2+) influx via NCX was increased by reducing [Na(+)]o to 13 mm. Low [Na(+)]o also increased the frequency of Ca(2+) waves and this effect was blocked by tetracaine and ryanodine but not 2-aminoethoxydiphenyl borate (2-APB). RT-PCR revealed that isolated ICC expressed both RyR2 and RyR3 subtypes. We conclude: (i) RyRs are required for the initiation of Ca(2+) waves, but wave propagation normally depends on activation of IP3 Rs; (ii) under resting conditions, propagation by IP3 Rs requires sensitisation by influx of Ca(2+) via reverse mode NCX; (iii) propagation can be maintained by RyRs if they have been sensitised to Ca(2+).
Assuntos
Sinalização do Cálcio , Cálcio/metabolismo , Células Intersticiais de Cajal/metabolismo , Potenciais de Ação , Animais , Células Cultivadas , Feminino , Células Intersticiais de Cajal/fisiologia , Masculino , Periodicidade , Coelhos , Canal de Liberação de Cálcio do Receptor de Rianodina/genética , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Trocador de Sódio e Cálcio/metabolismo , Uretra/citologiaRESUMO
Large conductance, voltage and Ca2+ activated K+ channels (BK channels) are abundantly expressed throughout the body and are important regulators of smooth muscle tone and neuronal excitability. Their dysfunction is implicated in various diseases including overactive bladder, hypertension and erectile dysfunction. Therefore, BK channel openers bear significant therapeutic potential to treat the above diseases. GoSlo-SR compounds were designed to be potent and efficacious BK channel openers. Although their structural activity relationships, activation in both BKα and BKαß channels and the hypothetical mode of action of these compounds has been studied in detail in recent years, their effectiveness to open the BKαγ channels still remains unexplored. In this study, we have examined the efficacy of 3 closely related GoSlo-SR openers, GoSlo-SR-5-6 (SR-5-6), GoSlo-SR-5-44 (SR-5-44) and GoSlo-SR-5-130 (SR-5-130) using inside out patches on BKα channels coexpressed with 4 different LRRC (γ1-4) subunits in HEK293 cells. Our data suggests that the activation effects due to SR-5-6 were not significantly affected in the presence of γ1-4 subunits. Interestingly, the effects of more efficacious BK channel opener SR-5-44 were altered by different γ subunits. In cells expressing BKα channels, the shift in V1/2 (ΔV1/2) induced by SR-5-44 (3 µM) was -76 ± 3 mV, whereas it was significantly reduced by â¼70 % in BKαγ1 channels (ΔV1/2= -23 ± 3, p < 0.001, ANOVA). In BKαγ2 channels the ΔV1/2 was -36 ± 1 mV, which was less than that observed in BKαγ3 and BKαγ4 channels where the ΔV1/2 was -47 ± 5 mV, and -82 ± 5 mV, respectively. Additionally, the excitatory effects of a 'ß specific' BK channel opener, SR-5-130 were only partially restored in the patches containing BKαγ1-4 channels. Together this data highlights that subtle modifications in GoSlo-SR structures alter their effectiveness on BK channels with accessory γ subunits and this study might provide a scaffold for the development of more tissue specific BK channel openers.
Assuntos
Antraquinonas/farmacologia , Canais de Potássio Ativados por Cálcio de Condutância Alta/fisiologia , Ácidos Sulfônicos/farmacologia , Células HEK293 , Humanos , Canais de Potássio Ativados por Cálcio de Condutância Alta/genética , Subunidades Proteicas/genética , Subunidades Proteicas/fisiologiaRESUMO
Interstitial cells of Cajal (ICC) have been identified in specific areas throughout the smooth musculature of the gastrointestinal (GI) tract. Located within the circular and longitudinal muscle layers of the gastric fundus lies a specific type of ICC, termed "intramuscular" ICC or IC-IM. The principal function of this cell type is to act as "mediators of excitatory and inhibitory enteric neurotransmission." The functional role of these cells has been investigated using W/W(v) mutant mice that specifically lack IC-IM, resulting in disrupted enteric neurotransmission. The aim of the present study was to investigate differential gene expression in W/W(v) mutant mice, from the tunica muscularis of the gastric fundus using a mouse cDNA microarray containing 1,081 known genes. Verification of the microarray data was attained using real-time "quantitative" PCR (qPCR). Of the 1,081 arrayed genes, 36 demonstrated differential expression by >2-fold in the W/W(v) mice. An agreement rate of 50% (7 of 14 tested) was obtained using qPCR. Of the seven confirmed changes in expression, several were indicative of a supersensitive phenotype, observed in denervation models. Expression of several putative neurotransmitter receptors including P2Y, the receptor for the inhibitory neurotransmitter ATP, was upregulated. The functional role of the P2Y receptor was also investigated using electrophysiological recordings. These results offer a new insight into the molecular changes that occur in W/W(v) fundic smooth muscle and may also provide novel information with regard to the importance of IC-IM in enteric neurotransmission.
Assuntos
Fundo Gástrico/citologia , Fundo Gástrico/metabolismo , Proteínas Proto-Oncogênicas c-kit/genética , Animais , Potenciais Evocados , Fundo Gástrico/fisiologia , Perfilação da Expressão Gênica , Camundongos , Camundongos Mutantes , Músculo Liso/citologia , Músculo Liso/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Fenótipo , Receptores Purinérgicos P2/fisiologia , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
We have designed, synthesised and characterised the effects of a number of novel anthraquinone derivatives and assessed their effects on large conductance, Ca(2+) activated K(+) (BK) channels recorded from rabbit bladder smooth muscle cells using the excised, inside/out configuration of the patch clamp technique. These compounds are members of the GoSlo-SR family of compounds, which potently open BK channels and shift the voltage required for half maximal activation (V1/2) negatively. The efficacy of the anilinoanthraquinone derivatives was enhanced when the size of ring D was increased, since the cyclopentane and cyclohexane derivatives shifted the V1/2, by -24 ± 6 mV and -54 ± 8 mV, respectively, whereas the cycloheptane and cyclooctane derivatives shifted the V1/2 by -61 ± 6 mV and -106 ± 6 mV. To examine if a combination of hydrophobicity and steric bulking of this region further enhanced their ability to open BK channels, we synthesised a number of naphthalene and tetrahydro-naphthalene derivatives. The tetrahydro-2-naphthalene derivative GoSlo-SR-5-69 was the most potent and efficacious of the series since it was able to shift the activation V1/2 by greater than -100 mV when applied at a concentration of 1 µM and had an EC50 of 251 nM, making it one of the most potent and efficacious BK channel openers synthesised to date.
Assuntos
Antraquinonas/química , Antraquinonas/farmacologia , Canais de Potássio Ativados por Cálcio de Condutância Alta/agonistas , Canais de Potássio Ativados por Cálcio de Condutância Alta/metabolismo , Músculo Liso/efeitos dos fármacos , Bexiga Urinária/efeitos dos fármacos , Animais , Antraquinonas/síntese química , Potenciais da Membrana/efeitos dos fármacos , Modelos Moleculares , Músculo Liso/fisiologia , Técnicas de Patch-Clamp , Coelhos , Bexiga Urinária/fisiologiaRESUMO
Opening up ion channels: We synthesised a series of anthraquinone analogues, called the GoSlo-SR family. Their effects on bladder smooth muscle BK channels were examined and, as shown, shifted voltage dependent activation >-100 mV (at 10 µM). They were more efficacious than NS11021 and could provide a new scaffold for the design of efficacious BK openers.