The sulphonylurea receptor SUR1 regulates ATP-sensitive mouse Kir6.2 K+ channels linked to the green fluorescent protein in human embryonic kidney cells (HEK 293).
1. Using a chimeric protein comprising the green fluorescent protein (GFP) linked to the C-terminus of the K+ channel protein mouse Kir6.2 (Kir6.2-C-GFP), the interactions between the sulphonylurea receptor SUR1 and Kir6.2 were investigated in transfected human embryonic kidney cells (HEK 293) by combined imaging and patch clamp techniques. 2. HEK 293 cells transfected with mouse Kir6.2-C-GFP and wild-type Kir6.2 exhibited functional K+ channels independently of SUR1. These channels were inhibited by ATP (IC50 = 150 microM), but were not responsive to stimulation by ADP or inhibition by sulphonylureas. Typically 15 +/- 7 active channels were found in an excised patch. 3. The distribution of Kir6.2-C-GFP protein was investigated by imaging of GFP fluorescence. There was a lamellar pattern of fluorescence labelling inside the cytoplasm (presumably associated with the endoplasmic reticulum and the Golgi apparatus) and intense punctate labelling near the cell membrane, but little fluorescence was associated with the plasma membrane. 4. In contrast, cells co-transfected with Kir6.2-C-GFP and SUR1 exhibited intense uniform plasma membrane labelling, and the lamellar and punctate labelling seen without SUR1 was no longer prominent. 5. In cells co-transfected with Kir6.2-C-GFP and SUR1, strong membrane labelling was associated with very high channel activity, with 484 +/- 311 active channels per excised patch. These K+ channels were sensitive to inhibition by ATP (IC50 = 17 microM), stimulated by ADP and inhibited by sulphonylureas. 6. We conclude that co-expression of SUR1 and Kir6.2 generates channels with the properties of native KATP channels. In addition, SUR1 promotes uniform insertion of Kir6.2-C-GFP into the plasma membrane and a 35-fold increase in channel activity, suggesting that SUR1 facilitates protein trafficking of Kir6.2 into the plasma membrane.
John
S A
SA
UCLA Cardiovascular Research Laboratory, Department of Medicine (Cardiology), UCLA School of Medicine 90095, USA.
Monck
J R
JR
Weiss
J N
JN
Ribalet
B
B
D000255:Adenosine Triphosphate / Q000502:physiology*; D000818:Animals; D002460:Cell Line; D004558:Electric Stimulation; D004594:Electrophysiology; D016695:Glycosyltransferases; D049452:Green Fluorescent Proteins; D006801:Humans; D007202:Indicators and Reagents; D007668:Kidney / Q000378:metabolism*; D008164:Luminescent Proteins; D008564:Membrane Potentials / Q000502:physiology; D008565:Membrane Proteins; D051379:Mice; D008856:Microscopy, Fluorescence; D018408:Patch-Clamp Techniques; D015221:Potassium Channels / Q000502:physiology*; D024661:Potassium Channels, Inwardly Rectifying; D012097:Repressor Proteins / Q000502:physiology*; D029701:Saccharomyces cerevisiae Proteins; D014162:Transfection
D007202:Indicators and Reagents; D008164:Luminescent Proteins; D008565:Membrane Proteins; D015221:Potassium Channels; D024661:Potassium Channels, Inwardly Rectifying; D012097:Repressor Proteins; D029701:Saccharomyces cerevisiae Proteins; D049452:Green Fluorescent Proteins; D000255:Adenosine Triphosphate; D016695:Glycosyltransferases; C092031:SUR1 protein, S cerevisiae