pH gating of ROMK (K(ir)1.1) channels: control by an Arg-Lys-Arg triad disrupted in antenatal Bartter syndrome

Abstract

Inward-rectifier K(+) channels of the ROMK (K(ir)1.1) subtype are responsible for K(+) secretion and control of NaCl absorption in the kidney. A hallmark of these channels is their gating by intracellular pH in the neutral range. Here we show that a lysine residue close to TM1, identified previously as a structural element required for pH-induced gating, is protonated at neutral pH and that this protonation drives pH gating in ROMK and other K(ir) channels. Such anomalous titration of this lysine residue (Lys-80 in K(ir)1.1) is accomplished by the tertiary structure of the K(ir) protein: two arginines in the distant N and C termini of the same subunit (Arg-41 and Arg-311 in K(ir)1.1) are located in close spatial proximity to the lysine allowing for electrostatic interactions that shift its pK(a) into the neutral pH range. Structural disturbance of this triad as a result from a number of point mutations found in patients with antenatal Bartter syndrome shifts the pK(a) of the lysine residue off the neutral pH range and results in channels permanently inactivated under physiological conditions. Thus, the results provide molecular understanding for normal pH gating of K(ir) channels as well as for the channel defects found in patients with antenatal Bartter syndrome.

Figure 1

pH gating of K_ir1.1 channels is driven by protonation of Lys-80 in the neutral pH range. (A) pH gating of K_ir1.1 channels measured in a giant inside-out patch from Xenopus oocytes on switching pH_i from 8.0 to 6.0 (inactivation) and from 6.0 to 8.0 (recovery from inactivation). Currents were recorded at a membrane potential of −80 mV, which was stepped up to 50 mV for 50 ms every 0.9 s. Solution application and changes in pH_i are illustrated by horizontal bars, time and current scales as indicated. (B) Same experiment as in A but with the pH-insensitive mutant K_ir1.1(K80M) in which Lys-80 was replaced by methionine. (C) (Upper) pH gating is abolished in K_ir1.1 channels by application of FmocCl at pH_i 7.5 before acidification. Changes in pH_i and application of FmocCl as indicated by horizontal bars. (Lower) Structure of FmocCl and its chemical reaction (nucleophilic substitution) with the amino group of a lysine residue.

Figure 2

Neutralization of Arg-41 and Arg-311 shifts titration of Lys-80 to the alkaline pH range by several pH units. (A) Cartoon summarizing the shift in pH_0.5 (ΔpH_0.5) observed on neutralization of the Arg/Lys residues in the N and C termini conserved among all K_ir proteins. Note that only R41Q and R311Q led to shifts in pH_0.5 (ΔpH_0.5) >3 pH units. (B) Patch recordings from oocytes expressing K_ir1.1(R41Q) (Upper) and K_ir1.1(R311Q) (Lower) at the pH_is indicated. Protocol as in Fig. 1. (C) Immunostaining of FLAG-tagged K_ir1.1(R311Q) and K_ir1.1 wild-type channels. Note staining of the plasma membrane in oocytes expressing either the K_ir1.1 wild-type or mutant subunit, whereas no staining was obtained in a control oocyte (water injected). (D) Rescue of channel activity by replacement of Lys-80 with methionine in R41Q, R311Q, and R41,311Q channels. Recordings as in A from K_ir1.1(R41Q,K80M), K_ir1.1(R311Q,K80M), and K_ir1.1(R41, 311Q,K80M) at the pH_is indicated.

Figure 3

Sensitivity of pH gating to side-chain variations at the C-terminal residue in K_ir4.1 and K_ir1.1 channels. (A and B) Shift in pH gating induced by neutralization of the C-terminal residue in K_ir4.1 channels. (A) Recordings from K_ir4.1 wild-type and K_ir4.1(R294Q) channels at the pH_is indicated; experimental protocol as in Fig. 1. (B) Current-pH_i relation of K_ir4.1(R294Q) and K_ir4.1 wild type. Note the shift in pH_0.5 observed for the mutant channels with respect to wild type [pH_0.5 and Hill coefficient were 9.5 and 1.4 for K_ir4.1(R294Q) and 6.1 and 2.1 for K_ir4.1 wild type]. (C) Effect of an Arg-Lys exchange at the C-terminal site in K_ir1.1 and K_ir4.1 channels on pH gating. Current-pH_i relation obtained from K_ir1.1 wild type and K_ir1.1(R311K) (squares) and from K_ir4.1 wild type and K_ir4.1(R294K) (circles). Lines represent fit of a Boltzmann function to the data (mean ± SD of eight and seven experiments for R311K and R294K, respectively). pH_0.5 and Hill coefficient, respectively, were 7.1 and 2.7 for K_ir1.1(R311K) and 8.0 and 1.7 for K_ir4.1(R294K).

Figure 4

Arg-Lys-Arg triad and gating is realized within one subunit. (A) (Upper) Cartoon illustrating the coexpression experiment (see text) designed to test for formation of the Arg-Lys-Arg triad via intersubunit interaction between Arg-41, Lys-80 [in K_ir1.1(R311Q, N171D)], and Arg-311 [in K_ir1.1(K80M)]. (Lower) Intersubunit interaction in a heteromultimeric complex assembled from three K_ir1.1(K80M) subunits and one K_ir1.1(R311Q, N171D) subunit. (B) Histograms illustrating current amplitudes (at −80 mV; Left), relative conductance at pH 8.4 in the presence of 100 μM SPM (at 50 mV; Center), and relative conductance at pH 6.0 in the absence of SPM (Right) observed with homomeric K_ir1.1(K80M) and channels forming on coexpression of K_ir1.1(K80M) and K_ir1.1(R311Q, N171D); all values are mean ± SD of 16–21 experiments (for the amplitudes) or of 8–11 experiments (for the conductances); relative conductance is normalization of conductance with respect to that observed in the absence of SPM or at a pH_i of 9.6. All coexpression experiments were done with an mRNA ratio of 1:10 (K80M: R311Q,N171D or K80M:N171D); note that all channels formed in the K80M:N171D coexpression were blocked by 100 μM SPM at 50 mV.

Figure 5

Disruption of the Arg-Lys-Arg triad as a result from mutations associated with aBS shifts pH gating off the neutral pH range. (A) Tertiary topology of the K_ir1.1 subunit as deduced here together with point mutations identified in patients with aBS to date. Filled circles represent aBS muations, open symbols represent residues of the Arg-Lys-Arg triad. Mutations (referred to the ROMK1 sequence) are C49Y, I51T, V72E, D74Y, W99C, D108H, P110L, V122E, N124K, G167E, L214V, S219R, L220F, R311Q/W, V315G, and F325C. (B) Current-pH_i relation obtained from K_ir1.1 wild type and the aBS mutations indicated. Lines represent fit of a Boltzmann function to the data (mean ± SD of 5–8 experiments); values for pH_0.5 were as follows: C49Y (7.5), I51T (7.3), D74Y (9.0), S219R (7.9), L220F (7.2), R311W (8.9), and V315G (6.9).