Korean J Physiol Pharmacol.  2003 Oct;7(5):267-273.

The Substates with Mutants That Negatively Charged Aspartate in Position 172 Was Replaced with Positive Charge in Murine Inward Rectifier Potassium Channel (Murine Kir2.1)

• So I
• Ashmole I
• Stanfield

Abstract

We have investigated the effect on inducing substate (s) of positively charged residues replaced in position 172 of the second transmembrane domain in murine inward rectifier potassium channels, formed by stable or transient transfection of Kir2.1 gene in MEL or CHO cells. Single channel recordings were obtained from either cell-attached patches or inside-out patches excised into solution containing 10 mM EDTA to rule out the effect of Mg2 on the channel gating. The substate (s) could be recorded with all mutants D172H, D172K and D172R. The unitary current-voltage (I-V) relation was not linear with D172H at pHi 6.3, whereas the unitary I-V relation was linear at pHi 8.0. The relative occupancy at SLC was increased from 0.018 at pHi 8.0 to 0.45 at pHi 5.5. In H-N dimer, that was increased from 0.016 at pHi 8.0 to 0.23 at pHi 5.5. The larger the size of the side chain or pKa with mutants (D172H, D172K and D172R), the more frequent the transitions between the fully open state and substate within an opening. The conductance of the substate also depended upon the pKa or the size of the side chain. The relative occupancy at substate SLC with monomer D172K (0.50) was less than that in K-H dimer (0.83). However, the relative occupancy at substate with D172R (0.79) was similar to that with R-N dimer (0.82). In the contrary to ROMK1, positive charge as well as negative charge in position 172 can induce the substate rather than block the pore in murine Kir2.1. The single channel properties of the mutant, that is, unitary I-V relation, the voltage dependence of the mean open time and relative occupancy of the substates and the increased latency to the first opening, explain the intrinsic gating observed in whole cell recordings.

Keyword

Potassium channel; Substate; Channel gating

MeSH Terms

Animals
Aspartic Acid*
CHO Cells
Cricetinae
Edetic Acid
Potassium Channels
Potassium Channels, Inwardly Rectifying*
Transfection
Aspartic Acid
Edetic Acid
Potassium Channels
Potassium Channels, Inwardly Rectifying