RESUMO
Inward rectifier potassium (Kir) channels are expressed in almost all mammalian tissues and contribute to a wide range of physiological processes. Kir4.1 channel expression is found in the brain, inner ear, eye, and kidney. Loss-of-function mutations in the pore-forming Kir4.1 subunit cause an autosomal recessive disorder characterized by epilepsy, ataxia, sensorineural deafness and tubulopathy (SeSAME/EST syndrome). Despite its importance in physiological and pathological conditions, pharmacological research of Kir4.1 is limited. Here, we characterized the effect of pentamidine on Kir4.1 channels using electrophysiology, mutagenesis and computational methods. Pentamidine potently inhibited Kir4.1 channels when applied to the cytoplasmic side under inside-out patch clamp configuration (IC50 = 97nM). The block was voltage dependent. Molecular modeling predicted the binding of pentamidine to the transmembrane pore region of Kir4.1 at aminoacids T127, T128 and E158. Mutation of each of these residues reduced the potency of pentamidine to block Kir4.1 channels. A pentamidine analog (PA-6) inhibited Kir4.1 with similar potency (IC50 = 132nM). Overall, this study shows that pentamidine blocks Kir4.1 channels interacting with threonine and glutamate residues in the transmembrane pore region. These results can be useful to design novel compounds with major potency and specificity over Kir4.1 channels.
Assuntos
Pentamidina/farmacologia , Bloqueadores dos Canais de Potássio/farmacologia , Canais de Potássio Corretores do Fluxo de Internalização/antagonistas & inibidores , Sítios de Ligação , Relação Dose-Resposta a Droga , Células HEK293 , Humanos , Interações Hidrofóbicas e Hidrofílicas , Simulação de Acoplamento Molecular , Pentamidina/metabolismo , Bloqueadores dos Canais de Potássio/química , Bloqueadores dos Canais de Potássio/metabolismo , Conformação ProteicaRESUMO
Inwardly rectifying potassium (Kir) channels are expressed in many cell types and contribute to a wide range of physiological processes. Particularly, Kir4.1 channels are involved in the astroglial spatial potassium buffering. In this work, we examined the effects of the cationic amphiphilic drug quinacrine on Kir4.1 channels heterologously expressed in HEK293 cells, employing the patch clamp technique. Quinacrine inhibited the currents of Kir4.1 channels in a concentration and voltage dependent manner. In inside-out patches, quinacrine inhibited Kir4.1 channels with an IC50 value of 1.8±0.3µM and with extremely slow blocking and unblocking kinetics. Molecular modeling combined with mutagenesis studies suggested that quinacrine blocks Kir4.1 by plugging the central cavity of the channels, stabilized by the residues E158 and T128. Overall, this study shows that quinacrine blocks Kir4.1 channels, which would be expected to impact the potassium transport in several tissues.
Assuntos
Canais de Potássio Corretores do Fluxo de Internalização/efeitos dos fármacos , Canais de Potássio Corretores do Fluxo de Internalização/metabolismo , Quinacrina/farmacologia , Animais , Astrócitos/metabolismo , Células HEK293 , Humanos , Ativação do Canal Iônico/fisiologia , Técnicas de Patch-Clamp/métodos , Potássio/metabolismo , Canais de Potássio/metabolismo , Canais de Potássio Corretores do Fluxo de Internalização/antagonistas & inibidores , Quinacrina/metabolismo , RatosRESUMO
Kir4.1 channels have been implicated in various physiological processes, mainly in the K+ homeostasis of the central nervous system and in the control of glial function and neuronal excitability. Even though, pharmacological research of these channels is very limited. Chloroquine (CQ) is an amino quinolone derivative known to inhibit Kir2.1 and Kir6.2 channels with different action mechanism and binding site. Here, we employed patch-clamp methods, mutagenesis analysis, and molecular modeling to characterize the molecular pharmacology of Kir4.1 inhibition by CQ. We found that this drug inhibits Kir4.1 channels heterologously expressed in HEK-293 cells. CQ produced a fast-onset voltage-dependent pore-blocking effect on these channels. In inside-out patches, CQ showed notable higher potency (IC50 ≈0.5µM at +50mV) and faster onset of block when compared to whole-cell configuration (IC50 ≈7µM at +60mV). Also, CQ showed a voltage-dependent unblock with repolarization. These results suggest that the drug directly blocks Kir4.1 channels by a pore-plugging mechanism. Moreover, we found that two residues (Thr128 and Glu158), facing the central cavity and located within the transmembrane pore, are particularly important structural determinants of CQ block. This evidence was similar to what was previously reported with Kir6.2, but distinct from the interaction site (cytoplasmic pore) CQ-Kir2.1. Thus, our findings highlight the diversity of interaction sites and mechanisms that underlie amino quinolone inhibition of Kir channels.