| Project/Area Number |
13558097
|
|---|
| Research Category |
Grant-in-Aid for Scientific Research (B)
|
| Allocation Type | Single-year Grants |
|---|
| Section | 展開研究 |
|---|
| Research Field |
神経・脳内生理学
|
|---|
| Research Institution | OKAZAKI NATIONAL RESEARCH INSTITUTES |
|---|
Principal Investigator |
NAKAI Junichi NATIONAL INSTITUTE FOR PHYSIOLOGICAL SCIENCES, DEPARTMENT OF IN FORMATION PHYSIOLOGY, RESEARCH ASSOCIATE, 生理学研究所, 助手 (80237198)
|
|---|
| Project Period (FY) |
2001 – 2002
|
| Project Status |
Completed (Fiscal Year 2002)
|
| Budget Amount *help |
¥11,700,000 (Direct Cost: ¥11,700,000)
Fiscal Year 2002: ¥2,300,000 (Direct Cost: ¥2,300,000)
Fiscal Year 2001: ¥9,400,000 (Direct Cost: ¥9,400,000)
|
|---|
| Keywords | Fluorescence / GFP / Potassium channel / Biosensor / Molecular biology / Membrane potential |
|---|
| Research Abstract |
Measurement of membrane voltage is one of the most important techniques to understand function of excitable cells. The purpose of this project is developing fluorescent protein probes which can sense membrane voltage, using Green Fluorescent Protein (GFP). To develop voltage-sensing fluorescent probes, one of the voltage-gated potassium channels were used as a base structure, and GFP was fused to the region near the S4 segment of this protein by means of molecular biological techniques. cDNAs were transfected to HEK293 cells and it was tested whether the fluorescenee from voltage-sensing probes change in response to the membrane voltage with the CCD camera system equipped on the fluorescent microscope. Unfortunately, the probes which had GFP near the C-terminal side of the S4 segment did not become fluorescent. Then, GFP was inserted at the N-terminus of S1, or the N-terminus of S3. These proteins became fluorescent. Parts of potassium channel (S1-S4, S3-S4, and S4-S6) were also used for fusion protein. All these truncated channels became fluorescent. One of the probes which used the S3-S4 region remained in cytoplasm and others was transported to the membrane. To improve the transportation of the proteins to the plasma membrane, the probes were fused to another protein which belongs to the ammo-acid transporters. The fused protein is transported to the plasma membrane better than the original protein. At this moment, the signals from the probes are so small to use as probes. We are trying to improve the signal/noise ration by changing the connecting sequences and the position of the GFP.
|
