2001 Fiscal Year Final Research Report Summary
Voltage-dependent gating and block by internal spermine in the inwardly rectifying K channels
| Project/Area Number |
11470013
|
|---|
| Research Category |
Grant-in-Aid for Scientific Research (B)
|
| Allocation Type | Single-year Grants |
|---|
| Section | 一般 |
|---|
| Research Field |
General physiology
|
|---|
| Research Institution | Kansai Medical University |
|---|
Principal Investigator |
MATSUDA Hiroko Kansai Medical University, Faculty of Medicine, Professor, 医学部, 教授 (10181736)
|
|---|
| Co-Investigator(Kenkyū-buntansha) |
OMORI Koicihro Kansai Medical University, Faculty of Medicine, Associate Professor, 医学部, 助教授 (80094465)
|
|---|
| Project Period (FY) |
1999 – 2001
|
| Keywords | K channel / Inward rectification / spermine |
|---|
| Research Abstract |
To investigate the mechanism of inward rectification, single-channel currents through inwardly rectifying K^+ (IRK1 ; Kir 2.1) channels were studied. cDNA encoding a wild-type (WT) IRK1 channel, D172N and tandem tetramer WT-(D172N)2-WT was transfected into COS-1 cells using the liposome method, and voltage clamp experiments were done after 48-72 h. Steady-state outward currents were recorded and open probability was calculated. The activation curve was fitted with single Boltzmann equation. The voltage of half-activation was 33.4 mV (WT), 51.2 mV (WT-(D172N)2-WT) and 76.6 mV (D172N). Open-and zero-current tirne histograms were constructed on current records at +42 mV The open-time histogram was fitted with a single exponential function. The mean open time was 53.1 ms (WT), 46.2 ms (WT-(D172N)2-WT) and 39.4 ms (D172N) in the control. Internal spermine reduced the open time in a concentration dependent manner: 37.8 ms (1 nM) and 11.7 ms (10 nM) in WT channels ; 34.7 ms (1 nM), 10.9 ms (10 nM) and 1.6 ms (100 nM) in WT-(D172N)2-WT channels ; 31.6 ms (1 nM), 11.7 ms (10 nM) and 1.7 ms (100 nM) in D172N channels. More than two exponential functions were necessary to fit the zero-cunent time histogram except in D172N channels with 10 and 100 nM spermine. These results suggest that intrinsic gating does exist separately from the spermine block. On the assumption of the state model (blocked state --- Open state --- closed state 1 --- closed state 2), blocking rates were estimatcd in each channel. They were 6.2-7.7 s^<-1> (1 nM), 64.7-70.1 s^<-1> (10 nM) and 563-603 s^<-1> (100 nM) and did not depend on the number of D172N mutant.
|
