Stabilization of the activity of ATP-sensitive potassium channels by ion pairs formed between adjacent Kir6.2 subunits

Abstract

ATP-sensitive potassium (KATP) channels are formed by the coassembly of four Kir6.2 subunits and four sulfonylurea receptor subunits (SUR). The cytoplasmic domains of Kir6.2 mediate channel gating by ATP, which closes the channel, and membrane phosphoinositides, which stabilize the open channel. Little is known, however, about the tertiary or quaternary structures of the domains that are responsible for these interactions. Here, we report that an ion pair between glutamate 229 and arginine 314 in the intracellular COOH terminus of Kir6.2 is critical for maintaining channel activity. Mutation of either residue to alanine induces inactivation, whereas charge reversal at positions 229 and 314 (E229R/R314E) abolishes inactivation and restores the wild-type channel phenotype. The close proximity of these two residues is demonstrated by disulfide bond formation between cysteine residues introduced at the two positions (E229C/R314C); disulfide bond formation abolishes inactivation and stabilizes the current. Using Kir6.2 tandem dimer constructs, we provide evidence that the ion pair likely forms by residues from two adjacent Kir6.2 subunits. We propose that the E229/R314 intersubunit ion pairs may contribute to a structural framework that facilitates the ability of other positively charged residues to interact with membrane phosphoinositides. Glutamate and arginine residues are found at homologous positions in many inward rectifier subunits, including the G-protein-activated inwardly rectifying potassium channel (GIRK), whose cytoplasmic domain structure has recently been solved. In the GIRK structure, the E229- and R314-corresponding residues are oriented in opposite directions in a single subunit such that in the tetramer model, the E229 equivalent residue from one subunit is in close proximity of the R314 equivalent residue from the adjacent subunit. The structure lends support to our findings in Kir6.2, and raises the possibility that a homologous ion pair may be involved in the gating of GIRKs.

Figure 1.

Identification of E229A as an inactivation mutant. (A) WT channel activities recorded in inside-out membrane patches using K-INT (left) or K-INT + 1 mM EDTA (right) bath solution. Arrows indicate patch excision. Channel rundown is dramatically reduced by the addition of 1 mM EDTA to the bath solution. (B) Using K-INT/EDTA solution, inactivation is observed in the E229A mutant channels. Representative currents recorded from inside-out membrane patches containing WT, the E229A, or the R314A mutant channels are superimposed for comparison. The arrow indicates patch excision into K-INT/EDTA. The kinetics of inactivation of the E229A channels are similar to the R314A channels. The inactivation time course of both E229A and R314A channels was fitted by a single exponential (indicated by the black curves). (C) Channels formed by WT-Kir6.2ΔC25 in the absence of SUR1 exhibited stable activities after patch excision into K-INT/EDTA (left). In contrast, channels formed by E229A-Kir6.2ΔC25 (middle) or R314A-Kir6.2ΔC25 (right) in the absence of SUR1 showed rapid current decay. Currents in this and subsequent figures were recorded at −50 mV membrane potential. Inward currents are shown as upward deflections.

Figure 2.

Channel inactivation can be recovered by exposure to ATP and subsequent washout of ATP. (A) Inactivated R314A (left) and E229A (right) channels were reactivated by exposure to ATP (5 mM) and subsequent washout of ATP. (B) A nonhydrolyzable ATP analogue, AMP-PNP mimicked the reactivation effect of ATP in both R314A (left) and E229A (right) channels. (C) The reactivation effect of ATP was not observed in channels formed by E229A-Kir6.2ΔC25 alone (left), but was retained in channels formed by E229A-Kir6.2ΔC25 in the presence of SUR1 (right).

Figure 3.

Inactivation of E229A mutant channels is reversed by PIP₂. Application of PIP₂ (10 μM) reversed inactivation and stimulated channel activity; it also decreased the ATP sensitivity of the channel, as was previously reported for WT channels (Baukrowitz et al., 1998; Shyng and Nichols, 1998).

Figure 4.

Open state stability is retained in the E229R/R314E double reverse mutant channels. (A) Representative currents recorded from inside-out membrane patches containing R192E, R301E, R314E, or E229R mutant channels. R301E channels did not give rise to measurable currents. All the other three mutant channels exhibited inactivation. (B) Currents recorded from membrane patches isolated from cells expressing the double charge reversal mutant E229R/R192E, E229R/R301E, or E229R/R314E. No currents were detected for the E229R/R301E channels. The E229R/R192E mutant channels still showed inactivation. In contrast, the E229R/R314E double mutant did not inactivate and behaved like WT channels.

Figure 5.

Characterization of the E229C and the R314C mutant channels. (A) A representative current trace recorded from an inside-out membrane patch containing the E229C mutant channels. The channels displayed inactivation similar to that observed in the E229A and E229R channels upon patch excision into the K-INT/EDTA solution. Exposure to 1 mM ATP (indicated by black bars below the recordings) partially reactivated the channels, which again underwent rapid inactivation. (B) The R314C Kir6.2 mutant protein is expressed and incorporated into the K_ATP channel complex. Surface immunofluorescent staining of cells coexpressing FLAG-tagged SUR1 and either the WT or the R314C mutant Kir6.2. Immunostaining was performed in living cells at 4°C using the M2 anti-FLAG monoclonal antibody followed by Cy3-conjugated secondary antibody. Surface labeling of the FLAG-SUR1 was observed in both cells coexpressing the WT or the R314C mutant Kir6.2, demonstrating that the R314C Kir6.2 is properly incorporated into the K_ATP channel complex.

Figure 6.

Disulfide bond formation between C229 and C314 in the E229C/R314C mutant channel stabilizes channel opening. Currents recorded from two separate membrane patches containing the E229C/R314C mutant channels. Upon patch excision into K-INT/EDTA, the channel exhibited low open probability and mild inactivation. However, the current gradually increased in amplitude and stability. Exposure of the patch to 5 mM ATP (indicated by the black bars underneath the current traces) accelerated the current reactivation process. (Top trace) Application of 1 mM DTT (in K-INT/EDTA, indicated by the gray bar) after current stabilization induced rapid inactivation of the channel in a reversible manner, indicating that the current stabilization was due to spontaneous disulfide bond formation. (Bottom trace) After the current has reached the maximum, application of 5 μM PIP₂ (see materials and methods) further stimulated channel activity by ∼2-fold.

Figure 7.

Disulfide bond formation that promotes channel opening in the E229C/R314C mutant does not involve endogenous cysteines. Representative recordings of WT and E229C channels showing the lack of time-dependent current increase in K-INT/EDTA and the lack of response to DTT.

Figure 8.

The E229/R314 ion pair is formed between two adjacent Kir6.2 subunits. (A) Currents recorded from a membrane patch containing Kir6.2 WT-WT (or ER-ER) dimer channels in the K-INT/EDTA bath solution. The dimer channel currents were stable and were inhibited by 5 mM ATP. Below the current trace is a cartoon illustrating the expected tetramer formed by the tandem dimer. The 229 residue in each subunit is shown in red and the 314 residue shown in blue. For each tandem dimer, the leading subunit is in gray and the trailing subunit in white. The expected ion pairs are connected by dotted lines. (B) In the EE-RR dimer channels, the possibility of ion pair formation in a single subunit is excluded. The channels behaved similarly to the WT-WT dimer channels; the rate of current decay is only slightly faster (see Table II). (C) In the AR-AR dimer channels, neither intra- nor intersubunit ion pair formation is possible. Currents from these channels decayed much faster than the WT-WT dimer channels, as expected. (D) In the RE-ER dimer channels, the possibility of ion pair formation between two adjacent subunits is excluded. Currents from these channels decayed much faster than the WT-WT, or the EE-RR dimer channels (see Table II).

Figure 9.

Proposed model for K_ATP channel inactivation. (A) Cartoon illustrating proposed physical relationships between the channel, ATP, and PIP₂ in the inactivation mutants. Disruption of the ion pair causes a structural change in the Kir6.2 tetramer, and possibly also a change in Kir6.2/SUR1 interactions, leading to channel inactivation. These structural changes can be overcome by increasing membrane PIP₂, or by ATP binding to the channel in a SUR1-dependent manner. Note that the four states presented should not be taken as detailed kinetic gating states of the channel, and that the transitions between the states are indicated only to illustrate the recovery effects of PIP₂ and ATP on inactivated channels. (B) Positions of the two ion pair-forming corresponding residues in the single (left) and tetramer (right) GIRK1 channel structures (Nishida and MacKinnon, 2002). Residue numbering in Kir6.2 is in black, and in GIRK1, red. The Kir6.2-E229 corresponding residue is shown as pink circles, and R314 corresponding residues as yellow circles.