Korean J Physiol Pharmacol.  2009 Feb;13(1):61-70. 10.4196/kjpp.2009.13.1.61.

Multiple Residues in the P-Region and M2 of Murine Kir 2.1 Regulate Blockage by External Ba2+

Affiliations
  • 1Department of Physiology and Biophysics, Seoul National University, College of Medicine, Seoul 110-799, Korea. stanfield@warwick.ac.uk, insuk@ plaza.snu.ac.kr
  • 2Department of Biological Sciences, University of Warwick, Coventry CV4 7AL; Ion Channel Group, UK.
  • 3Department of Cell Physiology & Pharmacology, University of Leicester, PO Box 138, Leicester, LE1 9HN, UK.
  • 4Department of Biochemistry, University of L:eicester, LE1 7RH, UK.

Abstract

We have examined the effects of certain mutations of the selectivity filter and of the membrane helix M2 on Ba2+ blockage of the inward rectifier potassium channel, Kir 2.1. We expressed mutant and wild type murine Kir 2.1 in Chinese hamster ovary (CHO) cells and used the whole cell patch-clamp technique to record K+ currents in the absence and presence of externally applied Ba2+. Wild type Kir2.1 was blocked by externally applied Ba2+ in a voltage and concentration dependent manner. Mutants of Y145 in the selectivity filter showed little change in the kinetics of Ba2+ blockage. The estimated Kd(0) was 108micrometer for Kir2.1 wild type, 124micrometer for a concatameric WT-Y145V dimer, 109micrometer for a WT-Y145L dimer, and 267micrometer for Y145F. Mutant channels T141A and S165L exhibit a reduced affinity together with a large reduction in the rate of blockage. In S165L, blockage proceeds with a double exponential time course, suggestive of more than one blocking site. The double mutation T141A/S165L dramatically reduced affinity for Ba2+, also showing two components with very different time courses. Mutants D172K and D172R (lining the central, aqueous cavity of the channel) showed both a decreased affinity to Ba2+ and a decrease in the on transition rate constant (kon). These results imply that residues stabilising the cytoplasmic end of the selectivity filter (T141, S165) and in the central cavity (D172) are major determinants of high affinity Ba2+ blockage in Kir 2.1.

Keyword

Potassium channel; Inward rectifier; Ionic selectivity; Barium blockage

MeSH Terms

Animals
Cricetinae
Cricetulus
Cytoplasm
Female
Kinetics
Membranes
Ovary
Patch-Clamp Techniques
Potassium Channels
Potassium Channels, Inwardly Rectifying
Potassium Channels
Potassium Channels, Inwardly Rectifying

Figure

  • Fig. 1. Ba2+ blockage of wild type Kir2.1 channels. (A) Membrane currents recorded from a single CHO cell expressing the gene for Kir2.1 in response to voltage steps from a holding potential of −17 mV to test potentials ranging from +63 mV to −117 mV, using 10 mV increments. Extracellular [K+] was 70 mM, and intracellular [K+] was 140 mM. (B~D) K+ currents activated by voltage steps from −17 mV to a range of potentials between +63 and −127 mV, in the presence of 10 μM (B), 100 μM (C), 1 mM (D) [Ba2+]o. The current was reduced to 3.8±0.6% of control levels after 1,500 ms at −117 mV in the presence of 100 μM Ba2+ (n=7).

  • Fig. 2. Voltage and concentration dependence of blockage by [Ba2+]o of Kir2.1. (A) Dose-response curves for extracellular Ba2+ blockage of steady-state IK at −57 mV (squares), −77 mV (triangles), −97 mV (inverted triangles), −117 mV (diamonds), −127 mV (circles). Lines are the best fits to the averaged data (n=7 for each point) using the Hill equation (Eqn. 1 of text). (B) The reciprocal of the time constant (τ) of exponential curves fitted to development of Ba2+ blockage plotted against Ba2+ concentration (n=5). Solid lines are linear fits to data using Eqn (3) of the text. Slopes (bottom to top) at −57 mV (squares), −77 mV (triangles), −97 mV (inverted triangles), −117 mV (diamonds), −127 mV (circles) were: 1.1×105 M−1s−1, 1.9×105 M−1s−1, 3.9×105 M−1s−1, 6.4×105 M−1s−1 and 9.1×105 M−1s−1, respectively.

  • Fig. 3. Blockage by Ba2+ is similar in Kir2.1 wild type, Y145F, WT-Y145L dimer and WT-Y145V dimer. (A) Plot of kon against membrane potential for wild type and Y145 mutants. kon values were obtained from the value of τ by fitting regression lines to Eqn (3). (B) Plot of koff against membrane potential for wild type and for Y145 mutants. koff values were obtained using Kd and kon. (C) Kd values obtained from the concentration-dependent inhibition of the steady-state currents were plotted against membrane potential. The solid line is the best fit of the data obtained at membrane potential (V) ranging from −127 mV to −47 mV, using the Woodhull equation (Eqn 4 of text).

  • Fig. 4. Ba2+ blockage of T141A. T141A slows block and lowers Ba2+ affinity (Kd(0)=1.02 mM; δ=0.61), primarily through a reduction in kon. (A, B) Blockage proceeds with a single exponential time course in both the wild type and mutant channel. Kir2.1 currents are shown for wild type (A) and T141A (B) in response to hyperpolarising pulses from a holding potential of −17 mV to voltages between −37 and −127 mV in 10 mV increments. (C) Relationship between the fractional steady state current (ordinate) and [Ba2+]o (abscissa) for wild type and T141A. (D) Relationship between the transition rate constants kon (above) and koff (below) plotted (ordinate) against membrane potential (abscissa) for wild type and T141A.

  • Fig. 5. Ba2+ blockage of S165L. S165L reduces Ba2+ affinity (Kd(0)=1.04 mM; δ=0.69) and slows blockage. With 300 μM Ba2+ the time-course of block becomes biphasic. The time course of each component and the relatively high affinity for Ba2+ makes detailed analysis of kinetics difficult.

  • Fig. 6. Ba2+ blockage of T141A/S165L. The double mutant T141A/S165L, which is permeable to Rb+ and Cs+, drastically reduces Ba2+ affinity, revealing two blocking sites. At high concentrations, blockage becomes dominated by the fast component allowing calculation of affinity and voltage- dependence (Kd(0)=210 mM; (δ=0.68). (A, B) Currents from T141A/S165L mutant channels obtained in the absence (A) and presence of 300 μM Ba2+ in response to hyperpolarising pulses from a holding potential of −17 mV to voltages between −37 and −127 mV. (C, D) The time constants obtained by fitting the decline in current with the sum of two exponentials and plotted (ordinates) against Ba2+ concentration for fast (C) and slow (D) components. (E) Plots of the transition rate constants (ordinate) kon (symbols in red) and koff (symbols in blue) plotted against membrane potential. kon for the faster component has the greater voltage dependence.

  • Fig. 7. Ba2+ blockage of Kir2.1 wild type and D172R. (A, B) Membrane currents recorded from a single CHO cell expressing the gene for wild type (A) and D172R (B) in response to voltage steps from a holding potential of −17 mV to test potentials ranging from +63 to −127 mV (10 mV increments) in the presence of 30 μM Ba2+. (C, D) Currents in wild type (C) and D172R (D) with 100μM Ba2+ at the voltage indicated. The time dependence of block fitted to a single exponential.

  • Fig. 8. D172K and D172R mutants decreased kon and increased the Kd. (A) Plot of kon against membrane potential for wild type (circles), D172R (squares), D172K (triangles). kon values were obtained from τ by fitting regression lines to Eqn (3) of the text. (B) Plot of koff against membrane potential for wild type (circles), D172R (squares) and D172K (triangles). koff values were obtained using Kd and kon. (C) Kd values obtained from the concentration-dependent inhibition of the steady-state currents were plotted against membrane potentials. The solid line is the best fit of the data obtained at membrane potential (V) ranging from −127 mV to −47 mV, using the Woodhull equation (Eqn 4 of the text). The values obtained for Kd(0) and δ are summarized in Table 1.


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