1998 Fiscal Year Final Research Report Summary
Molecular basis of the substate behavior in the inwardly rectifying K channels
| Project/Area Number |
09470013
|
|---|
| 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) |
YAMAMOTO Akitsugu Faculty of Medicine, Assistant Professor, 医学部, 講師 (30174775)
OMORI Koichiro Faculty of Medicine, Assistant Professor, 医学部, 助教授 (80094465)
|
|---|
| Project Period (FY) |
1997 – 1998
|
| Keywords | K channel / Inward rectification / IRK1 / Mg ion |
|---|
| Research Abstract |
To investigate tlie molecular basis of the sublevels induced in the outward current during block by internal Mg^<2+>, single-channel currents through inwardly rectifying K^+ (IRK1) channels were studied. cDNA encoding a functional murine IRK1 channel was transfected into COS-1 cells using the liposome method, and voltage clamp experiments were done after 48-72 h. Intracellular Mg^<2+>2 at micromolar concentrations induced sublevels in the outward current at one-third and two-thirds of the unitary amplitude in wild-type channels. Replacing Asp 172 with Asn (D172N) and Gln (0172Q) abolished these sublevels, i.e. the channel showed only the fully open and blocked states. Both mutations reduced the Mg^<2+> sensitivity of the channel at 2 muM Mg^<2+>. However, the Mg^<2+> sensitivity did not differsignificantly at higher concentrations (10 muM) and voltages (+70 mV), suggesting that a binding site other than D172 is critical in Mg^<2+> blockade. Channels expressed from D172E showed the sublevels, indicating that a negative charge is indispensable to the substate behavior. Channels from tandem tetramers of IRK1 with one and two D172N mutant subunits mainly showed sublevels with two-thirdsamplitude, while those from tetramers with three D172N mutant subunits showed no sublevels. These findings suggest that differences in Mg^<2+> binding patterns lead to different conductive states in a single-barrelled channel. A set of four aspartate carboxyates can interact simultaneously with two Mg^<2+> ions (this is possible only in wild-type channels), inducing the one-third level in the case where another binding site is free of Mg^<2+>2. If only one Mg^<2+> ion binds to the D172 site (this is possible in channels with D172 more than two) and another binding site is free, the two-thirds level appears.
|
