RESUMO
TREK channels produce background currents that regulate cell excitability. These channels are sensitive to a wide variety of stimuli including polyunsaturated fatty acids (PUFAs), phospholipids, mechanical stretch, and intracellular acidification. They are inhibited by neurotransmitters, hormones, and pharmacological agents such as the antidepressant fluoxetine. TREK1 knockout mice have impaired PUFA-mediated neuroprotection to ischemia, reduced sensitivity to volatile anesthetics, altered perception of pain, and a depression-resistant phenotype. Here, we investigate TREK1 regulation by Gq-coupled receptors (GqPCR) and phospholipids. Several reports indicate that the C-terminal domain of TREK1 is a key regulatory domain. We developed a fluorescent-based technique that monitors the plasma membrane association of the C terminus of TREK1 in real time. Our fluorescence and functional experiments link the modulation of TREK1 channel function by internal pH, phospholipid, and GqPCRs to TREK1-C-terminal domain association to the plasma membrane, where increased association results in greater activity. In keeping with this relation, inhibition of TREK1 current by fluoxetine is found to be accompanied by dissociation of the C-terminal domain from the membrane.
Assuntos
Membrana Celular/metabolismo , Fosfolipídeos/metabolismo , Canais de Potássio de Domínios Poros em Tandem/metabolismo , Animais , Antidepressivos de Segunda Geração/farmacologia , Membrana Celular/genética , Ácidos Graxos Insaturados/genética , Ácidos Graxos Insaturados/metabolismo , Fluoxetina/farmacologia , Células HEK293 , Humanos , Concentração de Íons de Hidrogênio , Isquemia/genética , Isquemia/metabolismo , Camundongos , Camundongos Knockout , Microscopia de Fluorescência , Oócitos , Fosfolipídeos/genética , Canais de Potássio de Domínios Poros em Tandem/genética , Estrutura Terciária de Proteína , Xenopus laevisRESUMO
In the voltage-sensing phosphatase Ci-VSP, a voltage-sensing domain (VSD) controls a lipid phosphatase domain (PD). The mechanism by which the domains are allosterically coupled is not well understood. Using an in vivo assay, we found that the interdomain linker that connects the VSD to the PD is essential for coupling the full-length protein. Biochemical assays showed that the linker is also needed for activity in the isolated PD. We also identified a late step of VSD motion in the full-length protein that depends on the linker. Notably, we found that this VSD motion requires PI(4,5)P2, a substrate of Ci-VSP. These results suggest that the voltage-driven motion of the VSD turns the enzyme on by rearranging the linker into an activated conformation, and that this activated conformation is stabilized by PI(4,5)P2. We propose that Ci-VSP activity is self-limited because its decrease of PI(4,5)P2 levels decouples the VSD from the enzyme.
Assuntos
Eletroquímica/métodos , Monoéster Fosfórico Hidrolases/química , Animais , Domínio Catalítico , Mutação , Monoéster Fosfórico Hidrolases/genética , Monoéster Fosfórico Hidrolases/metabolismoRESUMO
Potassium channels are among the core functional elements of life because they underpin essential cellular functions including excitability, homeostasis, and secretion. We present here a series of multivalent calix[4]arene ligands that bind to the surface of voltage-dependent potassium channels (K(v)1.x) in a reversible manner. Molecular modeling correctly predicts the best candidates with a conical C(4) symmetry for optimal binding, and the effects on channel function are assessed electrophysiologically. Reversible inhibition was observed, without noticeable damage of the oocytes, for tetraacylguanidinium or tetraarginine members of the series with small lower rim O-substituents. Apparent binding constants were in the low micromolar range and had Hill coefficients of 1, consistent with a single site of binding. Suppression of current amplitude was accompanied by a positive shift in the voltage dependence of gating and slowing of both voltage sensor motion and channel opening. These effects are in keeping with expectations for docking in the central pore and interaction with the pore domain "turret."
Assuntos
Calixarenos/química , Fenóis/química , Canais de Potássio de Abertura Dependente da Tensão da Membrana/química , Animais , Sítios de Ligação , Calixarenos/farmacologia , Cristalografia por Raios X , Relação Dose-Resposta a Droga , Eletrofisiologia , Feminino , Humanos , Cinética , Canal de Potássio Kv1.2/química , Canal de Potássio Kv1.2/genética , Canal de Potássio Kv1.2/fisiologia , Ligantes , Potenciais da Membrana/efeitos dos fármacos , Modelos Moleculares , Estrutura Molecular , Oócitos/efeitos dos fármacos , Oócitos/metabolismo , Oócitos/fisiologia , Fenóis/farmacologia , Canais de Potássio de Abertura Dependente da Tensão da Membrana/genética , Canais de Potássio de Abertura Dependente da Tensão da Membrana/fisiologia , Ligação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Xenopus laevisRESUMO
Voltage-sensing domains (VSDs) confer voltage dependence on effector domains of membrane proteins. Ion channels use four VSDs to control a gate in the pore domain, but in the recently discovered phosphatase Ci-VSP, the number of subunits has been unknown. Using single-molecule microscopy to count subunits and voltage clamp fluorometry to detect structural dynamics, we found Ci-VSP to be a monomer, which operates independently, but nevertheless undergoes multiple voltage-dependent conformational transitions.