The Korean Journal of Physiology and Pharmacology

The Korean Society of Pharmacology (대한약리학회)
권호 표지

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. (Ion Channel Group, Department of Cell Physiology and Pharmacology, University of Leicester,Department of Physiology and Biophysics, Seoul Notional University College of Medicine) ;
  • Ashmole, I. (Department of Biological Sciences, University of Warwick,Ion Channel Group, Department of Cell Physiology and Pharmacology, University of Leicester) ;
  • Stanfield, P.R. (Department of Biological Sciences, University of Warwick,Ion Channel Group, Department of Cell Physiology and Pharmacology, University of Leicester)
  • Published : 2003.10.21
Download PDF
⟨ Previous Next ⟩

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 $Mg^{2+}$ 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.

Keywords

References

  1. Chen X-H, Bezprozvanny I, Tsien R. Molecular basis of proton block of L-type Ca2$^+$ channels. J Gen Physiol 108: 363 374, 1996 https://doi.org/10.1085/jgp.108.5.363
  2. Dart C, Leyland ML, Barrett-Jolley R, Shelton PA, Spencer PJ, Conley EC, Sutcliffe MJ, Stanfield PR. The dependence of Ag$^+$ block of a potassium channel, murine Kir2.1, on a cysteine residue in the selectivity filter. J Physiol 511.1: 25 32, 1998
  3. Korchev YE, Bashford CL, Alder GM, Apel PY, Edmonds DT, Lev AA, Nandi K, Zima AV, Pasternak CA. A novel explanation for fluctuations of ion current through narrow pores. FASEB J 11: 600 608, 1997 https://doi.org/10.1096/fasebj.11.7.9212084
  4. Lu T, Nguyen B, Zhang X, Yang J. Architecture of a K$^+$ channel inner pore revealed by stoichiometric covalent modification. Neuron 22: 571 580, 1999 https://doi.org/10.1016/S0896-6273(00)80711-4
  5. Lu Z, MacKinnon R. Electrostatic tuning of Mg2$^+$ affinity in an inward-rectifier K$^+$ channel. Nature 371: 243 246, 1994 https://doi.org/10.1038/371243a0
  6. Lu Z, MacKinnon R. Probing a potassium channel pore with an engineered protonatable site. Biochemistry 34: 13133 13138, 1995 https://doi.org/10.1021/bi00040a026
  7. Oishi K, Omori K, Ohyama H, Shingu K, Matsuda H. Neutralization of aspartate residues in the murine inwardly rectifying K$^+$ channel IRK1 affects the substate behaviour in Mg2$^+$ block. J Physiol 510.3: 675 683, 1998
  8. Root MJ, MacKinnon R. Two identical noninteracting sites in an ion channel revealed by proton transfer. Science 265: 1852 1865, 1994 https://doi.org/10.1126/science.7522344
  9. Shyng SL, Ferrigni T, Nichols CG. Conrol of rectification and gating of cloned KATP channels by the Kir6.2 subunit. J Gen Physiol 110: 141 153, 1997 https://doi.org/10.1085/jgp.110.2.141
  10. So I, Ashmole I, Soh H, Park CS, Spencer PJ, Leyalnd M, Stanfield PR. Instrinsic gating in inward rectifier potassium channels (Kir2.1) with low polyamine affinity generated by site directed mutagenesis. Kor J Physiol Pharmacol 7: 131 142, 2003
  11. So I, Ashmole I, Stanfield PR. Conductance substates in mutants of the murine inward rectifier potassium channel Kir2.1. Biophys J 78: 2032Pos, 2000
  12. Stanfield PR, Davies NW, Shelton PA, Khan IA, Brammer WJ, Standen NB, Conley EC. The intrinsic gating of inward rectifier K$^+$ channels expressed from the murine IRK1 gene depends on voltage, K$^+$ and Mg$^{2+}$. J Physiol 475: 1-7, 1994 https://doi.org/10.1113/jphysiol.1994.sp020044