RESUMO
Many applications utilizing artificial lipid bilayers require the ability to exchange the bilayer's solution environment. However, because of the instability of the bilayer, the rate of solution exchange is limited, which significantly hinders the measurement rate and throughput. We have developed an artificial bilayer system that can withstand high flow speeds, up to 2.1 m/s, by supporting the bilayer with a hydrogel. We demonstrated the ability to measure during flow by measuring the conductance of gramicidin-A channels while switching between solutions of two different compositions, recording a time to measure 90% change in current of approximately 2.7 seconds at a flow rate of 0.1 m/s. We also demonstrated a potential application of this system by measuring the conductance modulation of the rat TRPM8 ion channel by an agonist and antagonist at varying concentrations, obtaining 7-point IC50 and EC50 values in approximately 7 minutes and 4-point values within 4 minutes.
Assuntos
Hidrogel de Polietilenoglicol-Dimetacrilato/química , Bicamadas Lipídicas/química , Soluções/química , Animais , Compostos de Boro/química , Compostos de Boro/metabolismo , Condutividade Elétrica , Gramicidina/química , Lipossomos/química , Lipossomos/metabolismo , Potenciais da Membrana , Mentol/química , Mentol/metabolismo , Ratos , Canais de Cátion TRPM/agonistas , Canais de Cátion TRPM/antagonistas & inibidores , Canais de Cátion TRPM/metabolismo , Fatores de TempoRESUMO
We show measurements of the human cardiac potassium ion channel Kv11.1 (hERG) in droplet bilayers incorporated directly from commercial membrane preparations of HEK293 cells. Although we do not obtain ensemble conductance kinetics and rectification observed in patch clamp measurements of hERG, ensemble currents measured in our system showed inhibition dependent on astemizole and E-4031 concentration, with IC50 values similar to those found with patch clamp. The availability of engineered HEK cells expressing a variety of ion channels, combined with the simplicity of the inhibition measurement, suggest that droplet bilayers may have considerable technological potential for determination of ion channel drug potency.
Assuntos
Avaliação Pré-Clínica de Medicamentos/instrumentação , Canais de Potássio Éter-A-Go-Go/química , Bicamadas Lipídicas/química , Potenciais da Membrana/efeitos dos fármacos , Técnicas Analíticas Microfluídicas/instrumentação , Bloqueadores dos Canais de Potássio/química , Bloqueadores dos Canais de Potássio/farmacologia , Canal de Potássio ERG1 , Desenho de Equipamento , Análise de Falha de Equipamento , Canais de Potássio Éter-A-Go-Go/efeitos dos fármacos , Ativação do Canal Iônico/efeitos dos fármacosRESUMO
The potency of pharmaceutical compounds acting on ion channels can be determined through measurements of ion channel conductance as a function of compound concentration. We have developed an artificial lipid bilayer chip for simple, fast, and high-yield measurement of ion channel conductance with simultaneous solution perfusion. Here we show the application of this chip to the measurement of the mammalian cold and menthol receptor TRPM8. Ensemble measurements of TRPM8 as a function of concentration of menthol and 2-aminoethoxydiphenyl borate (2-APB) enabled efficient determination of menthol's EC(50) (111.8 µM ± 2.4 µM) and 2-APB's IC(50) (4.9 µM ± 0.2 µM) in agreement with published values. This validation, coupled with the compatibility of this platform with automation and parallelization, indicates significant potential for large-scale pharmaceutical ion channel screening.
