RESUMO
The ability to apply large-scale screening formats to measures of ion channel function offers immense opportunities for drug discovery and academic research. Technologies have been developed over the last several years that now provide the ability to screen large numbers of compounds and natural products on ion channel function to find novel drugs. Application of these technologies has vastly improved the capabilities of ion channel drug discovery and provides an avenue to accelerate discoveries of ion channel biology. These advances have largely arisen from the development and application of instruments and reporters of membrane potential and ion movements in cells used to measure functional activity of ion channels. This article endeavors to describe the practical applications of these technologies in developing, validating, and implementing high throughput screening assay formats to different types of ion channels.
Assuntos
Bioquímica/métodos , Biotecnologia/métodos , Proteínas de Ligação ao GTP/química , Canais Iônicos/química , Receptores de Superfície Celular/química , Sítio Alostérico , Animais , Automação , Sítios de Ligação , Desenho de Fármacos , Humanos , Íons , Ligantes , Potenciais da Membrana , Ligação ProteicaRESUMO
A variety of transfection approaches have been used to deliver plasmids encoding ion channel genes into cells. We have used the baculovirus transduction system, BacMam, to demonstrate transient expression of multi-subunit KATP channels in CHO-K1 and HEK-293 EBNA cells using sulfonylurea receptor 1 (SUR), SUR2A, SUR2B, and KIR 6.2 genes. [3H]-glyburide binding, patch clamp, and DiBAC4(3) measurements of membrane potential changes were used to monitor channel expression. BacMam delivery of each SUR isoform with KIR6.2 was demonstrated based on its pharmacological profiles. Expression levels of SUR1 and KIR6.2 were titrated by varying the viral concentration or time of virus addition, with functional activity measured in as little as 4-6 hours posttransduction. Further increases in BacMam virus induced sufficient KATP expression to dominate membrane potential without pharmacological opening of the channel. Independently altering treatment with virus containing either the SUR1 or KIR6.2 gene revealed interactions among subunits during formation of functional channels in the plasma membrane. This study demonstrates the utility and versatility of BacMam as a valuable gene delivery tool for the study of ion channel function.
Assuntos
Transportadores de Cassetes de Ligação de ATP , Canais de Potássio Corretores do Fluxo de Internalização/genética , Canais de Potássio Corretores do Fluxo de Internalização/metabolismo , Canais de Potássio/genética , Canais de Potássio/metabolismo , Receptores de Droga/genética , Receptores de Droga/metabolismo , Animais , Baculoviridae/genética , Células CHO , Linhagem Celular , Cricetinae , Expressão Gênica , Glibureto/metabolismo , Humanos , Potenciais da Membrana , Plasmídeos/genética , Canais de Potássio/química , Canais de Potássio Corretores do Fluxo de Internalização/química , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores de Droga/química , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Receptores de Sulfonilureias , Transdução GenéticaRESUMO
A series of 7-substituted-3-cyclobutylamino-4H-1,2,4-benzothiadiazine-1,1-dioxide derivatives has been synthesized and evaluated as K(ATP) channel agonists using the inside-out excised patch clamp technique. The most active compounds were approximately 20-fold more potent than diazoxide in opening K(ATP) channels. A linear relationship exists between the potency of the compound and the sigma value of the 7-substituent with electron-withdrawing groups exhibiting higher activity. These compounds may be useful in modulating insulin release from pancreatic beta-cells and in diseases associated with hyperinsulinemia.