Stabilization of ion selectivity filter by pore loop ion pairs in an inwardly rectifying potassium channel

Abstract

Ion selectivity is critical for the biological functions of voltage-dependent cation channels and is achieved by specific ion binding to a pore region called the selectivity filter. In voltage-gated K+, Na+ and Ca2+ channels, the selectivity filter is formed by a short polypeptide loop (called the H5 or P region) between the fifth and sixth transmembrane segments, donated by each of the four subunits or internal homologous domains forming the channel. While mutagenesis studies on voltage-gated K+ channels have begun to shed light on the structural organization of this pore region, little is known about the physical and chemical interactions that maintain the structural stability of the selectivity filter. Here we show that in an inwardly rectifying K+ (IRK) channel, IRK1, short range interactions of an ion pair in the H5 pore loop are crucial for pore structure and ion permeation. The two residues, a glutamate and an arginine, appear to form exposed salt bridges in the tetrameric channel. Alteration or disruption of such ion pair interactions dramatically alters ion selectivity and permeation. Since this ion pair is conserved in all IRK channels, it may constitute a general mechanism for maintaining the stability of the pore structure in this channel superfamily.

Figure 1

Functional interactions between a pair of pore loop residues. (a) Alignment of the H5 pore loop amino acid sequences from members of six subfamilies of inwardly rectifying K⁺ channels. See Doupnik et al. (3) for nomenclature. The IRK1 channel used in this study corresponds to Kir2.1. The amino acid sequence corresponds to number 132–148. The H5 region sequence of the voltage-gated Shaker K⁺ channel is also shown for comparison. (b–d) Whole-cell currents recorded by two-electrode voltage-clamp from oocytes injected with cRNA for E138R (b), R148E (c), or E138R/R148E (d). Currents were elicited by 200-ms voltage steps to −100 to +40 mV in 20-mV increments from a holding potential of −60 mV and are not leak-subtracted. The dashed line indicates zero current level. The current amplitude at −100 mV was 111 ± 7 nA (mean ± SEM, n = 11) for E138R, 119 ± 7 nA (n = 11) for R148E, and 671 ± 27 nA (n = 20) for E138R/R148E. Oocytes injected with cRNAs for E138D, E138Q, E138C, R148K, R148H, or R148C, like oocytes injected with cRNAs for E138R or R148E, produced currents not significantly different from uninjected oocytes, which had a basal current of 116 ± 21 nA (n = 21) at −100 mV. (e–f) Current–voltage relations in 90 mM external KCl or NaCl for the wild-type IRK1 channel or mutant E138R/R148E channel. Current was measured at the end of a 200-ms test pulse and was leak-subtracted. For the wild-type channel, the reversal potential was −5.9 ± 0.3 mV (mean ± SEM, n = 9) in KCl and −103 ± 3 mV (n = 9) in NaCl. For the mutant channel, the reversal potential was −4.8 ± 1.0 mV (n = 11) in KCl and −7.2 ± 1.2 mV (n = 11) in NaCl.

Figure 2

pH titration suggests formation of Glu-138–Arg-148 salt bridges. (a) The carboxyl group of Glu-138 and the guanido group of Arg-148 are proposed to form a salt bridge, which involves both short range electrostatic attraction and hydrogen bonding. (b) Effect of external pH on whole-cell current produced by the wild-type IRK1 channel, recorded by two-electrode voltage-clamp. Each data point is an average of 5–8 measurements. Solid line represents a least-squares fit to the Hill equation with a pK_a of 1.97 and a Hill coefficient of 4.1.

Figure 3

Mutation of Glu-138 or Arg-148 alters pH titration. (a) Effect of external pH on whole-cell current recorded by two-electrode voltage-clamp from oocytes expressing the WT·E138Q·E138Q·WT or WT·R148H·R148H·WT tetramer. Data for the wild-type channel are also shown for comparison. Each data point is an average of 5–8 measurements. Solid lines represent least-squares fit of the data for each channel type (see text for detail). (b) A close-up of the response in the acidic pH range.

Figure 4

Disruption of one or two Glu-138–Arg-148 ion pairs alters ion permeation. (Left) Cell-attached single-channel currents recorded at −120 mV from oocytes expressing the wild-type channel or mutant channels generated by the WT·E138D·WT·WT or WT·E138D· E138D·WT tetramer. (Center) The corresponding all point amplitude histogram. The presumed channel type is depicted on the Right.