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

Young Mee Lee; Gareth A. Thompson; Ian Ashmole; Mark Leyland; Insuk So; Peter R. Stanfield

doi:10.4196/kjpp.2009.13.1.61

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Lee, Thompson, Ashmole, Leyland, So, and Stanfield: Multiple Residues in the P-Region and M2 of Murine Kir 2.1 Regulate Blockage by External Ba2+

Original Article

Korean J Physiol Pharmacol 2009;13(1):61-70.

Published online: 17 February 2009

DOI: https://doi.org/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⁺

Young Mee Lee^1,^†, Gareth A. Thompson^3,^†, Ian Ashmole², Mark Leyland⁴, Insuk So^1,^∗, Peter R. Stanfield²

¹Department of Physiology and Biophysics, Seoul National University, College of Medicine, Seoul 110-799, Korea

²Department of Biological Sciences, University of Warwick, Coventry CV4 7AL; Ion Channel Group, PO Box 138, Leicester, LE1 9HN, 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

^∗Corresponding to: Insuk So, Department of Physiology and Biophysics, Seoul National University, College of Medicine, 28 Yonggon-dong, Chongno-gu, Seoul 110-799, Korea. (Tel) 82-2-740-8228, (Fax) 82-2-763-9667, (E-mail) p.r.stanfield@warwick.ac.uk or insuk@plaza.snu.ac.kr

^†Co-Corresponding author: YM Lee and GA Thompson contributed equally to this work and should be considered as joint first author.

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

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 K_d(0) was 108 μM for Kir2.1 wild type, 124 μM for a concatameric WT-Y145V dimer, 109 μM for a WT-Y145L dimer, and 267 μM 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 (k_on). 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.

Keywords: Potassium channel, Inward rectifier, Ionic selectivity, Barium blockage

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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 I_K 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 k_on against membrane potential for wild type and Y145 mutants. k_on values were obtained from the value of τ by fitting regression lines to Eqn (3). (B) Plot of k_off against membrane potential for wild type and for Y145 mutants. k_off values were obtained using K_d and k_on. (C) K_d 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 (K_d(0)=1.02 mM; δ=0.61), primarily through a reduction in k_on. (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 k_on (above) and k_off (below) plotted (ordinate) against membrane potential (abscissa) for wild type and T141A.

Fig. 5.

Ba²⁺ blockage of S165L. S165L reduces Ba²⁺ affinity (K_d(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 (K_d(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) k_on (symbols in red) and k_off (symbols in blue) plotted against membrane potential. k_on 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 k_on and increased the K_d. (A) Plot of k_on against membrane potential for wild type (circles), D172R (squares), D172K (triangles). k_on values were obtained from τ by fitting regression lines to Eqn (3) of the text. (B) Plot of k_off against membrane potential for wild type (circles), D172R (squares) and D172K (triangles). k_off values were obtained using K_d and k_on. (C) K_d 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 K_d(0) and δ are summarized in Table 1.

Table 1.

Ba²⁺ blockage in wild type and in mutant channels

Channel mutant	K_d(0) (μM)	δ	k_on (−127 mV) (M⁻¹s⁻¹)	k_off (−127 mV) (s⁻¹)
Kir2.1-WT	108	0.43	9.1×10⁵	1.35
WT-Y145V dimer	124	0.43	1.0×10⁶	1.89
WT-Y145L dimer	109	0.40	8.9×10⁵	2.85
Y145F	267	0.51	5.9×10⁵	1.10
T141A	858	0.57	9.5×10⁴
S165L	1,040	0.69	1.6×10⁴
T141A/S165L	473,000	1.08
D172K	2,500	0.42	1.3×10⁴	0.54
D172R	1,800	0.49	6.4×10⁴	0.91

K_d(0) is the dissociation constant at 0 mV and δ is the electrical distance between the outside of the membrane and the blocking site, both obtained using Eqn (4) of the text. The values for the transition rate constants k_on and k_off are derived using Eqn (3) of the text and are given here for a membrane potential of −127 mV.

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