2004 Fiscal Year Final Research Report Summary
Simultaneous measurement of ATP-sensitive K current and fluorescent imaging.
| Project/Area Number |
14370012
|
|---|
| Research Category |
Grant-in-Aid for Scientific Research (B)
|
| Allocation Type | Single-year Grants |
|---|
| Section | 一般 |
|---|
| Research Field |
General physiology
|
|---|
| Research Institution | JICHI MEDICAL University (2004)
Kyoto University (2002-2003) |
|---|
Principal Investigator |
TAKANO Makoto JICHI MEDICAL University, School of Medicine, Department of Physiology, Professor, 医学部, 教授 (30236252)
|
|---|
| Project Period (FY) |
2002 – 2004
|
| Keywords | Kir6.2 / SUR2A |
|---|
| Research Abstract |
The ATP-sensitive K^+ (K_<ATP>) channel is a heteromultimer composed of four inwardly rectifying K^+ channels (Kir6.2) and four sulfonylurea receptors (SUR). Intracellular ATP binds to Kir6.2 and inhibits K_<ATP> channel. Nucleotide diphosphates (NDPs) binds to SUR, thereby increases the open probability of, and decreases the ATP-sensitivity of K_<ATP> channel. SUR is a member of ATP-binding cassette (ABC) superfamily. possessing two nucleotide binding folds (NBF). Prokaryotic members of ABC superfamily such as HisP and RbsA are termed "half-size ABC proteins", and possess only one NBF, and form a functional dimer in the cell membrane. We splited SUR2A into two "half-size molecules" before and after NDB1, and found that they could reassemble with Kir6.2 to form a functional K_<ATP> channel. C-terminal half SUR (C640) conferred glibenclamide sensitivity to Kir delta C36, whereas C640 did not increase open probability of Kir6.2 delta C36. We also made fusion proteins of yellow fluorescent protein (YFP) or cyan fluorescent protein (CFP) with split SURs and Kir6.2, and tried to identify fluorescent resonance energy transfer (FRET) between CYP and YFP. However, pharmacological stimuli with K channel openers and glibenclamide failed to induce the changes of FRET ratio. We also found that fluorescent labeled ATP could successfully inhibited the fusion protein of GFP and Kir6.2delta C36. However, it was difficult to identify FRET between fluorescent labeled ATP and GFP.
|
