RESUMO
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
A collagenase-proteinase mixture was used to isolate airway smooth muscle cells (ASMC) from rabbit bronchi, and membrane currents were recorded using the whole cell patch-clamp technique. Stepping from -100 mV to a test potential of -40 mV evoked a fast voltage-dependent Na(+) current, sometimes with an amplitude of several nanoamperes. The current disappeared within 15 min of exposure to papain + DTT (n = 6). Comparison of the current in ASMC with current mediated by NaV1.5 α-subunits expressed in human embryonic kidney cells revealed similar voltage dependences of activation (V1/2 = -42 mV for NaV1.5) and sensitivities to TTX (IC50 = 1.1 and 1.2 µM for ASMC and NaV1.5, respectively). The current in ASMC was also blocked by lidocaine (IC50 = 160 µM). Although veratridine, an agonist of voltage-gated Na(+) channels, reduced the peak current by 33%, it slowed inactivation, resulting in a fourfold increase in sustained current (measured at 25 ms after onset). In current-clamp mode, veratridine prolonged evoked action potentials from 37 ± 9 to 1,053 ± 410 ms (n = 8). Primers for NaV1.2-1.9 were used to amplify mRNA from groups of â¼20 isolated ASMC and from whole bronchial tissue by RT-PCR. Transcripts for NaV1.2, NaV1.3, and NaV1.5-1.9 were detected in whole tissue, but only NaV1.2 and NaV1.5 were detected in single cells. We conclude that freshly dispersed rabbit ASMC express a fast voltage-gated Na(+) current that is mediated mainly by the NaV1.5 subtype.
Assuntos
Brônquios/metabolismo , Miócitos de Músculo Liso/metabolismo , Canal de Sódio Disparado por Voltagem NAV1.5/metabolismo , Animais , Brônquios/citologia , Brônquios/efeitos dos fármacos , Separação Celular/métodos , Regulação da Expressão Gênica , Células HEK293 , Humanos , Ativação do Canal Iônico , Cinética , Potenciais da Membrana , Canal de Sódio Disparado por Voltagem NAV1.5/efeitos dos fármacos , Canal de Sódio Disparado por Voltagem NAV1.5/genética , Técnicas de Patch-Clamp , RNA Mensageiro/metabolismo , Coelhos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Bloqueadores dos Canais de Sódio/farmacologia , TransfecçãoRESUMO
The homodimeric muscle isoform of creatine kinase (MM-CK) unfolds on exposure to low levels of guanidinium chloride (GdmCl) to yield a partly folded monomeric intermediate. Those regions of MM-CK that experience local unfolding were previously identified through an extensive study of antibody accessibility and protease sensitivity. Since these studies were completed, the coordinates of the rabbit isoform (MM-CK) were released. In light of this, we have determined the minimum changes to this structure required to explain our data on protease and epitope accessibility in the intermediate. We propose that the observed changes occur through (a) disruption of the monomer-monomer interface during dissociation, (b) separation and/ or unfolding of domains or subdomains, and (c) the partial unfolding of solvent-exposed helices. The proposed structure for the intermediate is consistent both with current models of unfolding intermediates and the results of independent studies pertaining to the unfolding of creatine kinase. Proteins 2001;42:269-278.