• The Korean Journal of Physiology and Pharmacology
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• v.7 no.5
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• pp.267-273
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• 2003

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 $Mg^{2+}$ 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 $pH_i$ 6.3, whereas the unitary I-V relation was linear at $pH_i$ 8.0. The relative occupancy at $S_{LC}$ was increased from 0.018 at $pH_i$ 8.0 to 0.45 at $pH_i$ 5.5. In H-N dimer, that was increased from 0.016 at $pH_i$ 8.0 to 0.23 at $pH_i$ 5.5. The larger the size of the side chain or $pK_a$ 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 $S_{LC}$ 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.

• The Korean Journal of Physiology and Pharmacology
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• v.2 no.6
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• pp.705-714
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• 1998

Cerebral blood vessels relax when extracellular $K^+$ concentrations $([K^+])_e$ are elevated moderately $(2{\sim}15$ mM, $K^+-induced$ vasorelaxation). We have therefore studied the underlying mechanism for this $K^+-induced$ vasorelaxation in the isolated middle cerebral arteries (MCAs). The effects of ouabain and $Ba^{2+}\;on\;K^+-induced$ vasorelaxation were examined to determine the role of sodium pump and/or Ba-sensitive process (possibly, inward rectifier K current) in the mechanism. Mulvany myograph was used to study 24 rats, 18 rabbits, and 10 humans MCAs $(216{\pm}3\;{\mu}m,\;347{\pm}7\;{\mu}m,\;and\;597{\pm}39\;{\mu}m$ in diameter when stretched to a tension equivalent to 55 mmHg). High $K^+$ (125 mM) and $PGF_{2{\alpha}}\;(1{\sim}10\;{\mu}M)$ induced concentration-dependent contractions in all 3 species, while histamine $(10{\sim}50\;{\mu}M)$ evoked contraction only in the rabbits and induced relaxation in the rats and humans. Addition of $K^+\;(2{\sim}10\;{\mu}M)$ to the control solution induced vasorelaxations. These effects were inhibited by the pretreatment with both ouabain $(10\;{\mu}M)$ and $Ba^{2+}\;(0.1{\sim}0.3\;mM)$ in the rat, but only with ouabain $(10\;{\mu}M)$ in the rabbit and human. These results suggest that $K^+-induced$ vasorelaxation occurs via the stimulation of electrogenic Na pump in the rabbit and human MCAs, while in the rat MCAs via the activation of both Na pump and Ba-sensitive process.