Forced gating motions by a substituted titratable side chain at the bundle crossing of a potassium channel

Abstract

Numerous inwardly rectifying potassium (Kir) channels possess an aromatic residue in the helix bundle crossing region, forming the narrowest pore constriction in crystal structures. However, the role of the Kir channel bundle crossing as a functional gate remains uncertain. We report a unique phenotype of Kir6.2 channels mutated to encode glutamate at this position (F168E). Despite a prediction of four glutamates in close proximity, Kir6.2(F168E) channels are predominantly closed at physiological pH, whereas alkalization causes rapid and reversible channel activation. These findings suggest that F168E glutamates are uncharged at physiological pH but become deprotonated at alkaline pH, forcing channel opening due to mutual repulsion of nearby negatively charged side chains. The potassium channel pore scaffold likely brings these glutamates close together, causing a significant pK(a) shift relative to the free side chain (as seen in the KcsA selectivity filter). Alkalization also shifts the apparent ATP sensitivity of the channel, indicating that forced motion of the bundle crossing is coupled to the ATP-binding site and may resemble conformational changes involved in wild-type Kir6.2 gating. The study demonstrates a novel mechanism for engineering extrinsic control of channel gating by pH and shows that conformational changes in the bundle crossing region are involved in ligand-dependent gating of Kir channels.

FIGURE 1.

Reverse pH dependence of Kir6.2(F168E) channels. A, structure of the KirBac1.1 channel, with bundle crossing residue Phe-146 highlighted in red. B, alignment of multiple Kir channel M2 segments. The first amino acid corresponds to the putative “glycine hinge” residue. The highlighted position corresponds to the aromatic bundle crossing residue depicted in A. C and D, continuous inside-out patch clamp recordings of WT Kir6.2 and Kir6.2(F168E) (both coexpressed with SUR1 in COSm6 cells), respectively, with internal pH changes as indicated. A small concentration of Ba²⁺ was included in solutions to assess leak with brief pulses to +50 mV.

FIGURE 2.

Hypothetical mechanism for pH dependence of Kir6.2(F168E) channels. A and B, carboxylate side chains are forced into close proximity by the channel pore scaffold and may undergo a significant pK_a shift. At sufficiently high pH, the carboxylate side chains will be deprotonated, resulting in mutual repulsion that forces motion around the helix bundle crossing. C, close arrangement of carboxylate side chains in the selectivity filters of KcsA and KirBac1.1. This orientation likely results in a substantial pK_a shift of carboxylates.

FIGURE 3.

Subunit stoichiometry of F168E effects on pH-dependent gating. A–D, tandem-linked channel tetramers containing one (FFFE; D), two (FFEE and FEFE; B and C, respectively), or three copies (FEEE; A) of the F168E mutant were characterized for their pH dependence. Channels with more than one F168E subunit exhibited activation at alkaline pH. E and F, representative inside-out patches from Xenopus oocytes expressing Kir6.2(F168E)ΔC36 and WT Kir6.2ΔC36 in the absence of SUR1 exhibit pH responses similar to those of the full-length channel. G, summary data for tetrameric channels with composition indicated by F (for WT subunit) or E (for F168E subunit). Data are also included for a WT Kir6.2 + F168E dimeric channel ('FE'+'FE') (n = 5 for FEEE, FFEE, FEFE, and FFFE tetramers; n = 4 for the FE dimer; n = 10 for WT Kir6.2ΔC36; and n = 11 for Kir6.2(F168E)ΔC36).

FIGURE 4.

Microscopic effects of pH-dependent activation of Kir6.2(F168E) channels. A, continuous record at −50 mV of a tetrameric (FEEE) construct (three copies of Kir6.2(F168E) + one copy of WT Kir6.2), with pH jumps between 6.5 and 8.5 as indicated. Downward deflections at pH changes are solution exchange artifacts and were minimized during figure preparation. The FEEE tetramer was used because it exhibited similar pH dependence as F168E channels but took considerably longer to express currents and was very amenable to recording single channel currents. B, expanded view of channel openings in regions i, ii, and iii indicated in A. Significant changes in single channel current magnitude were not observed, but openings were far more frequent at alkaline pH. Similar observations were made in four patches.

FIGURE 5.

Position-specific requirements for alkalization-dependent activation of Kir6.2. Glutamate mutants at inner cavity-lining positions were tested for pH dependence. Representative currents at −50 mV with pH jumps as indicated are presented for each pore-lining mutant channel. Only Kir6.2(F168E) channels (boldface arrow, lower right) exhibited activation at alkaline pH.

FIGURE 6.

Neutralization of titratable residues near F168E. A, molecular model of the bundle crossing region of Kir6.2. B–E, double mutants Kir6.2(H70A/F168E) (n = 7), Kir6.2(F168E/K170A) (n = 7), and Kir6.2(F168E/H175A) (n = 5) were examined for pH dependence. All double mutants exhibited alkalization-dependent activation comparable with Kir6.2(F168E) channels.

FIGURE 7.

Surface expression of Kir6.2 bundle crossing mutants. A, Western blots for FLAG-SUR1 in cells transfected with FLAG-SUR1 + WT Kir6.2, Kir6.2(F168E), or Kir6.2(F168D) or in the absence of any Kir6.2 subunit. Two bands were typically observed in cells transfected with FLAG-SUR1 together with a channel construct. Only the lower molecular weight band was apparent when FLAG-SUR1 was transfected alone. A longer exposure is also presented for Kir6.2(F168E) + SUR1 (boxed), which typically exhibited lower intensity bands relative to other transfections. The arrow indicates the higher molecular weight band corresponding to mature glycosylated FLAG-SUR1. B, Western blot for FLAG-SUR1 in cells transfected with WT Kir6.2 + FLAG-SUR1. Purification of surface-biotinylated protein resulted in a single immunoreactive band corresponding to the mature glycosylated form of FLAG-SUR1 (indicated by the arrow), whereas the entire cell lysate generated both the core and mature glycosylated forms.

FIGURE 8.

Open state stability mutations affect the phenotype of Kir6.2(F168E) channels. Representative inside-out patch records are presented for Kir6.2(F168E/I296L) (A) and Kir6.2(F168E/R176A) (B) channels, with pH jumps as indicated. C, summary data for both mutant channels (n = 4 for I296L and n = 7 for R176A).

FIGURE 9.

Alkalization-dependent activation alters ATP inhibition of Kir6.2(F168E) channels. A, continuous records at −50 mV for a patch expressing WT Kir6.2 + SUR1, with ATP concentration jumps as indicated. Raw current records were extracted from the same patch and normalized to the peak current in 0 ATP at either pH 7.3 (gray) or 8.3 (black). B, summary of ATP dose response for WT Kir6.2 at pH 7.3 (n = 8), 8.3 (n = 4), and 8.8 (n = 4). Irel is the ratio of current in ATP to the control current level (0 ATP) at a given pH. C and D, identical experiments and summarized data as in A and B for Kir6.2(F168E) channels (n = 10 for pH 7.3 and n = 5 for pH 8.3 and 8.8). Dose-response curves were fit with the equation Irel = (1 − C)/(1 + ([ATP]/K_d)^h) + C. E, schematic model illustrating state preference for ATP binding and pH-dependent changes in open state stability.

FIGURE 10.

ATP inhibition does not cause complete closure of Kir6.2(F168E) channels. A, ATP inhibition of Kir6.2(F168E) channels was measured at pH 7.3, resulting in a clear plateau conductance that persisted even at 5 mm ATP, with considerably reduced current fluctuations relative to the 0 ATP condition. The presence of a plateau conductance was validated by blocking currents completely with a pulse from −50 to +50 mV in the presence of spermine. B, currents of similar magnitude through WT Kir6.2 channels exhibited virtually complete inhibition by ATP.