Titration of KATP channel expression in mammalian cells utilizing recombinant baculovirus transduction.
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.
Pfohl
Jeffrey L
JL
Department of Systems Research, GlaxoSmithKline, 5 Moore Drive, Research Triangle Park, NC 27709, USA.
Worley
Jennings F
JF
Condreay
J Patrick
JP
An
Gang
G
Apolito
Christopher J
CJ
Kost
Tom A
TA
Truax
James F
JF
D018528:ATP-Binding Cassette Transporters; D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D016466:CHO Cells; D002460:Cell Line; D006224:Cricetinae; D015870:Gene Expression; D005905:Glyburide / Q000378:metabolism; D006801:Humans; D008564:Membrane Potentials; D010957:Plasmids / Q000235:genetics; D015221:Potassium Channels / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D024661:Potassium Channels, Inwardly Rectifying / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D011955:Receptors, Drug / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D064233:Sulfonylurea Receptors; D014161:Transduction, Genetic
C577507:ABCC8 protein, human; C577510:ABCC9 protein, human; D018528:ATP-Binding Cassette Transporters; D015221:Potassium Channels; D024661:Potassium Channels, Inwardly Rectifying; D012333:RNA, Messenger; D011955:Receptors, Drug; D011994:Recombinant Proteins; D064233:Sulfonylurea Receptors; D005905:Glyburide