RESUMO
Mechanosensitive channels are present in almost every living cell, yet the evidence for their functional presence in T lymphocytes is absent. In this study, by means of the patch-clamp technique in attached and inside-out modes, we have characterized cationic channels, rapidly activated by membrane stretch in Jurkat T lymphoblasts. The half-activation was achieved at a negative pressure of ~50mm Hg. In attached mode, single channel currents displayed an inward rectification and the unitary conductance of ~40 pS at zero command voltage. In excised inside-out patches the rectification was transformed to an outward one. Mechanosensitive channels weakly discriminated between mono- and divalent cations (PCa/PNa~1) and were equally permeable for Ca²âº and Mg²âº. Pharmacological analysis showed that the mechanosensitive channels were potently blocked by amiloride (1mM) and Gd³âº (10 µM) in a voltage-dependent manner. They were also almost completely blocked by ruthenium red (1 µM) and SKF 96365 (250 µM), inhibitors of transient receptor potential vanilloid 2 (TRPV2) channels. At the same time, the channels were insensitive to 2-aminoethoxydiphenyl borate (2-APB, 100 µM) or N-(p-amylcinnamoyl)anthranilic acid (ACA, 50 µM), antagonists of transient receptor potential canonical (TRPC) or transient receptor potential melastatin (TRPM) channels, respectively. Human TRPV2 siRNA virtually abolished the stretch-activated current. TRPV2 are channels with multifaceted functions and regulatory mechanisms, with potentially important roles in the lymphocyte Ca²âº signaling. Implications of their regulation by mechanical stress are discussed in the context of lymphoid cells functions.
Assuntos
Cálcio/metabolismo , Ativação do Canal Iônico/fisiologia , Mecanotransdução Celular/fisiologia , Canais de Cátion TRPV/metabolismo , Amilorida/farmacologia , Compostos de Boro/farmacologia , Expressão Gênica , Humanos , Imidazóis/farmacologia , Transporte de Íons/efeitos dos fármacos , Transporte de Íons/fisiologia , Células Jurkat , Leucemia de Células T/genética , Leucemia de Células T/metabolismo , Leucemia de Células T/fisiopatologia , Magnésio/metabolismo , Potenciais da Membrana/genética , Potenciais da Membrana/fisiologia , Técnicas de Patch-Clamp , Potássio/metabolismo , Interferência de RNA , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Rutênio Vermelho/farmacologia , Sódio/metabolismo , Canais de Cátion TRPV/antagonistas & inibidores , Canais de Cátion TRPV/genéticaRESUMO
Recent studies have shown the potential application of ivermectins in the treatment of alcohol use disorder (AUD). Ivermectin is a positive allosteric modulator (PAM) of P2X4R and this molecule exerts its action in the transmembrane region (known as the TM region) of trimeric channel structure (the pocket formed by Asp331, Met336, Trp46, Trp50, and Tyr42). The aim of this study is to identify FDA drugs with potential PAM properties, by exploring the P2X4Rs from four organisms (Danio rerio, Mus musculus, Rattus norvegicus, and Homo sapiens). The in silico study consists of carrying out the molecular docking of 1656 FDA-approved drugs on the structure of P2X4R, using the commercially available compounds from the ZINC15 database for virtual screening. To strengthen the reliability of the results, two docking protocols were used involving the use of two programs, Autodock 4.2 and Autodock Vina. Nine FDA drugs with potential PAM properties were identified. In addition, eight molecules with potential negative allosteric modulator (NAM) action, and 13 molecules with potential allosteric modulator (AM) action were identified. The FDA drugs identified in this study with PAM, NAM, and AM action, shared in the P2X4Rs of the four organisms, can provide a guideline to proceed with research concerning new drugs for the study and treatment of AUD.
Assuntos
Alcoolismo/tratamento farmacológico , Antagonistas do Receptor Purinérgico P2X/farmacologia , Receptores Purinérgicos P2X4/efeitos dos fármacos , Regulação Alostérica , Sequência de Aminoácidos , Animais , Simulação por Computador , Aprovação de Drogas , Humanos , Camundongos , Modelos Moleculares , Simulação de Acoplamento Molecular , Conformação Proteica , Antagonistas do Receptor Purinérgico P2X/uso terapêutico , Ratos , Receptores Purinérgicos P2X4/química , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Especificidade da Espécie , Estados Unidos , United States Food and Drug Administration , Peixe-ZebraRESUMO
The aim of the present study was to investigate if P2X4 receptors are expressed in murine myenteric neurons and if these receptors contribute to form functional channels in the neuronal membrane by using molecular and electrophysiological techniques. The whole-cell recording technique was used to measure membrane currents induced by ATP (I(ATP)) in myenteric neurons. Compared with recombinant P2X4 receptor-channels (reported by others in a previous study), native myenteric P2X receptors have a relative lower sensitivity for ATP (EC50=102 µM) and α,ß methylene ATP (not effect at 30 or 100 µM). BzATP was a weak agonist for native P2X receptors. KN-62 had no effect on myenteric P2X channels whereas PPADS (IC50=0.54 µM) or suramin (IC50=134 µM) were more potent antagonists than on P2X4 homomeric channels. I(ATP) were potentiated by ivermectin (effect that is specific on P2X4 receptors) and zinc. Western blotting shows the presence of P2X4 protein and RT-PCR the corresponding mRNA transcript in the small intestine. Immunoreactivity for P2X4 receptors was found in most myenteric neurons in culture. Single-cell RT-PCR shows the presence of P2X4 mRNA in 90% of myenteric neurons. Our results indicate that P2X4 receptors are expressed in the majority of myenteric neurons, contribute to the membrane currents activated by ATP, and because most properties of I(ATP) does not correspond to P2X4 homomeric channels it is proposed that P2X4 are forming heteromeric channels in these neurons. P2X4 subunits have a widespread distribution within the myenteric plexus and would be expected to play an important role in cell signaling.