RESUMEN
Large conductance, calcium- and voltage-gated potassium (BK) channels are ubiquitous and critical for neuronal function, immunity, and smooth muscle contractility. BK channels are thought to be regulated by phosphatidylinositol 4,5-bisphosphate (PIP(2)) only through phospholipase C (PLC)-generated PIP(2) metabolites that target Ca(2+) stores and protein kinase C and, eventually, the BK channel. Here, we report that PIP(2) activates BK channels independently of PIP(2) metabolites. PIP(2) enhances Ca(2+)-driven gating and alters both open and closed channel distributions without affecting voltage gating and unitary conductance. Recovery from activation was strongly dependent on PIP(2) acyl chain length, with channels exposed to water-soluble diC4 and diC8 showing much faster recovery than those exposed to PIP(2) (diC16). The PIP(2)-channel interaction requires negative charge and the inositol moiety in the phospholipid headgroup, and the sequence RKK in the S6-S7 cytosolic linker of the BK channel-forming (cbv1) subunit. PIP(2)-induced activation is drastically potentiated by accessory beta(1) (but not beta(4)) channel subunits. Moreover, PIP(2) robustly activates BK channels in vascular myocytes, where beta(1) subunits are abundantly expressed, but not in skeletal myocytes, where these subunits are barely detectable. These data demonstrate that the final PIP(2) effect is determined by channel accessory subunits, and such mechanism is subunit specific. In HEK293 cells, cotransfection of cbv1+beta(1) and PI4-kinaseIIalpha robustly activates BK channels, suggesting a role for endogenous PIP(2) in modulating channel activity. Indeed, in membrane patches excised from vascular myocytes, BK channel activity runs down and Mg-ATP recovers it, this recovery being abolished by PIP(2) antibodies applied to the cytosolic membrane surface. Moreover, in intact arterial myocytes under physiological conditions, PLC inhibition on top of blockade of downstream signaling leads to drastic BK channel activation. Finally, pharmacological treatment that raises PIP(2) levels and activates BK channels dilates de-endothelized arteries that regulate cerebral blood flow. These data indicate that endogenous PIP(2) directly activates vascular myocyte BK channels to control vascular tone.
Asunto(s)
Activación del Canal Iónico/fisiología , Canales de Potasio de Gran Conductancia Activados por el Calcio/fisiología , Fosfatidilinositol 4,5-Difosfato/fisiología , Transducción de Señal/fisiología , Adenosina Trifosfato/farmacología , Animales , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/farmacología , Calcio/farmacología , Electrofisiología , Inhibidores Enzimáticos/farmacología , Femenino , Subunidades alfa de los Canales de Potasio de Gran Conductancia Activados por Calcio/genética , Subunidades alfa de los Canales de Potasio de Gran Conductancia Activados por Calcio/fisiología , Canales de Potasio de Gran Conductancia Activados por el Calcio/antagonistas & inhibidores , Arteria Cerebral Media/citología , Arteria Cerebral Media/efectos de los fármacos , Arteria Cerebral Media/fisiología , Modelos Biológicos , Miocitos del Músculo Liso/efectos de los fármacos , Miocitos del Músculo Liso/fisiología , Ácido Ocadaico/farmacología , Oocitos/efectos de los fármacos , Oocitos/metabolismo , Oocitos/fisiología , Fosfatidilinositol 4,5-Difosfato/análogos & derivados , Fosfatidilinositol 4,5-Difosfato/farmacología , Fosfatos de Fosfatidilinositol/farmacología , Fosfatidilinositoles/farmacología , Polilisina/farmacología , ARN Complementario/genética , Ratas , Ratas Sprague-Dawley , Transducción de Señal/efectos de los fármacos , Xenopus laevisRESUMEN
High-conductance, Ca(2+)-activated and voltage-gated (BK) channels set neuronal firing. They are almost universally activated by alcohol, leading to reduced neuronal excitability and neuropeptide release and to motor intoxication. However, several BK channels are inhibited by alcohol, and most other voltage-gated K(+) channels are refractory to drug action. BK channels are homotetramers (encoded by Slo1) that possess a unique transmembrane segment (S0), leading to a cytosolic S0-S1 loop. We identified Thr107 of bovine slo (bslo) in this loop as a critical residue that determines BK channel responses to alcohol. In addition, the activity of Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) in the cell controlled channel activity and alcohol modulation. Incremental CaMKII-mediated phosphorylation of Thr107 in the BK tetramer progressively increased channel activity and gradually switched the channel alcohol responses from robust activation to inhibition. Thus, CaMKII phosphorylation of slo Thr107 works as a 'molecular dimmer switch' that could mediate tolerance to alcohol, a form of neuronal plasticity.