A Conserved Residue Cluster That Governs Kinetics of ATP-dependent Gating of Kir6.2 Potassium Channels

Abstract

ATP-sensitive potassium (KATP) channels are heteromultimeric complexes of an inwardly rectifying Kir channel (Kir6.x) and sulfonylurea receptors. Their regulation by intracellular ATP and ADP generates electrical signals in response to changes in cellular metabolism. We investigated channel elements that control the kinetics of ATP-dependent regulation of KATP (Kir6.2 + SUR1) channels using rapid concentration jumps. WT Kir6.2 channels re-open after rapid washout of ATP with a time constant of ∼60 ms. Extending similar kinetic measurements to numerous mutants revealed fairly modest effects on gating kinetics despite significant changes in ATP sensitivity and open probability. However, we identified a pair of highly conserved neighboring amino acids (Trp-68 and Lys-170) that control the rate of channel opening and inhibition in response to ATP. Paradoxically, mutations of Trp-68 or Lys-170 markedly slow the kinetics of channel opening (500 and 700 ms for W68L and K170N, respectively), while increasing channel open probability. Examining the functional effects of these residues using φ value analysis revealed a steep negative slope. This finding implies that these residues play a role in lowering the transition state energy barrier between open and closed channel states. Using unnatural amino acid incorporation, we demonstrate the requirement for a planar amino acid at Kir6.2 position 68 for normal channel gating, which is potentially necessary to localize the ϵ-amine of Lys-170 in the phosphatidylinositol 4,5-bisphosphate-binding site. Overall, our findings identify a discrete pair of highly conserved residues with an essential role for controlling gating kinetics of Kir channels.

Keywords: KATP channel; PIP2; diabetes; gating; ion channel; kinetics; ligand-dependent gating; potassium channel.

FIGURE 1.

Rapid solution exchange of inside-out patches expressing K_ATP channel mutants. A, using a rapid perfusion apparatus, a patch expressing K_ATP channels was switched between 50 mm K⁺ and 150 mm K⁺ solutions. The change in driving force causes a change in magnitude of ionic current, which is used to evaluate the speed of solution exchange. B, exemplar current traces depicting ATP concentration jumps applied to WT K_ATP channels (Kir6.2 + SUR1) and various mutants, using inside out patch recordings with a membrane potential of −50 mV (inward currents are depicted in the positive/upward direction and have been normalized to illustrate the time course of channel reopening). Inset shows noise levels of WT and C166S patches with equal current magnitudes. C, location of residues in the bundle crossing and domain interface region that were tested for kinetics of reactivation after ATP washout. D–G, plots of reactivation kinetics after ATP washout, together with an equilibrium constant derived from stationary noise analysis (see under “Materials and Methods”). For each position studied, n values were as follows: C166: S(6), L(6), I(5), T(5), V(5); Q52: A(5), R(5); G53: A(7), D(13); V59: A(5).

FIGURE 2.

Rapid solution exchange reveals a rate-controlling position at Trp-68 for channel reopening. A, kinetics of channel reopening were examined for a family of mutations at position Trp-68 in Kir6.2 channels, using inside-out patches at a membrane voltage of −50 mV (inward currents are depicted in the positive/upward direction and are normalized to illustrate the time course of channel reopening). Profound slowing of channel reopening was observed. The inset panel depicts macroscopic currents from similarly sized current traces from WT Kir6.2 (black) or W68L mutant (red), illustrating lower macroscopic noise in W68L mutant channels. B, reopening rates are plotted against an equilibrium constant estimated from stationary noise analysis. For each mutant, n-values were as follows: F(7), D(7), E(5), L(6), A(8). C, sequence alignment of multiple Kir channels illustrates strong conservation of residue Trp-68.

FIGURE 3.

Lys-170 mutations recapitulate the effects of Trp-68 mutations. A–C, reopening rates are plotted against an equilibrium constant derived from stationary noise analysis for three residues (Ile-167, Lys-170, and Thr-171) in close proximity to Trp-68. For each position, n-values were as follows: I167: L(6), F(6); T171: A(5), S(6); K170: N(7), E(7), S(6), Q(5), R(6). D, exemplar current traces depicting channel reopening kinetics for mutations at position Lys-170, using inside-out patches at a membrane voltage of −50 mV (inward currents are depicted in the positive/upward direction and are normalized to illustrate the time course of channel reopening). Marked slowing of channel reopening is apparent. E, sequence alignment of multiple Kir channels illustrates strong conservation of a Lys at position 170. F, depiction of the three residues in close proximity to Trp-68 in a model Kir6.2 structure based on Protein Data Bank code 3SYA.

FIGURE 4.

Parameters of ATP sensitivity and channel function in Trp-68 and Lys-170 mutants. A and B, inhibition of current relative to control was determined for a range of ATP concentrations applied to inside-out patches from indicated Trp-68 and Lys-170 mutant channels (n values are in parentheses in the symbol legend). C, current variance and mean macroscopic current are presented for numerous cells transfected with each indicated mutant (n values were 8 (WT), 7 (W68A), 6 (W68L), 7 (K170N), 7 (K170Q)). Note the consistently higher variance in WT Kir6.2 relative to channel mutants across a range of current magnitudes.

FIGURE 5.

Large multiple sequence alignment of Kir channels. A, depiction of a sequence alignment of Kir channels (based on the Pfam Kir channel database), with the % identity plotted for each position. B, subset of residue positions with greater than 90% identity is highlighted (gray bar in A), including Kir6.2 position Trp-68. Position Lys-170 just fails to meet the 90% threshold in the alignment. C, structural depiction of highly conserved residues on the 3SYA structure (Kir3.2). Highlighted residues are clustered into regions contributing to the selectivity filter (yellow), the core of the cytoplasmic domain (orange), or two highly conserved clusters at the cytoplasmic domain:transmembrane domain interface (green and blue).

FIGURE 6.

Slow channel reactivation reflects a slow conformational change and not slow ligand unbinding. A–C, rapid solution exchanges were used to determine kinetics of reopening after washout of ATP, ADP, and AMP in Kir6.2 (A), Kir6.2(K170N) (B), and Kir6.2(W68L) (C) mutants. D, kinetics of channel reopening did not vary significantly between nucleotides for each mutant, and slow gating kinetics persisted in the K170N and W68L mutants (n = 5–6 patches per experimental condition). E, scheme depicting fast unbinding of ATP, followed by a slow conformational rearrangement whose rate is controlled by the Trp-68/Lys-170 cluster.

FIGURE 7.

Prolonged burst and interburst intervals in Kir6.2(K170N) mutant channels. A, single channel currents over a short time scale illustrating rapid intraburst kinetics of WT Kir6.2 and Kir6.2(K170N) channels. B, closed time histogram illustrating the frequency of events of short duration closures (shorter than ∼200 ms) for both WT Kir6.2 (2719 closures in three patches) and Kir6.2(K170N) channels (1851 closures in three patches). C and D, histograms illustrating the duration of channel closures lasting 10 ms or longer (interbursts), and duration of bursts of openings (delineated by closures lasting longer than 10 ms). For WT patches, proportions were calculated for 425 burst/interburst events recorded in three patches. For Kir6.2(K170N) patches, bursting events were much longer (often 30 s or more), and interbursts were much less frequent, so proportions are illustrated for 64 burst/interburst events observed in three patches. E, representative single channel currents in symmetrical 140 mm K⁺ solutions with a membrane potential of −80 mV.

FIGURE 8.

Expression of Kir6.2 channels with nonsense suppression for unnatural amino acid mutagenesis. A, schematic diagram of unnatural amino acid approach for incorporation of Trp analogs at Kir6.2 position 68. B, whole oocyte currents under two-electrode voltage clamp from oocytes co-injected with WT Kir6.2 + SUR1, with voltage ramps between −80 and +80 mV. Channels were activated using 3 mm azide for metabolic inhibition and inhibited with the sulfonylurea glibenclamide. C, no glibenclamide-sensitive currents were observed in oocytes injected with Kir6.2(Trp-68TAG) + SUR1 + pdCpA (unloaded tRNA control). D, large glibenclamide-sensitive currents could be elicited in oocytes co-injected with Kir6.2(Trp-68TAG) + SUR1 + aminoacylated tRNA (sample currents are presented using F₄-Trp rescue of Kir6.2(Trp-68TAG)).

FIGURE 9.

Unnatural amino acid mutagenesis demonstrates the importance of a planar amino acid at position Trp-68. A–D, unnatural amino acid mutagenesis was used to introduce subtle Trp variants at Kir6.2 position 68. Chemical structures and the kinetics of channel reactivation after jumps from 1 mm ATP into 0 ATP solutions are depicted, using inside-out patches at a membrane voltage of −50 mV (inward currents are depicted in the positive/upward direction and are normalized to illustrate the time course of channel reopening). E, plot of reopening rates (k_open) versus equilibrium constant (K_eq) for unnatural mutations at position Trp-68 (colored symbols). Data from Fig. 2 have also been included for comparison (black symbols). For each mutant, n values were as follows: F₄-Trp(6), indole (Ind)(6), cyclohexylalanine (CHA)(5), WT Trp(5). F, mean rates of reactivation for the unnatural amino acid substitutions at Kir6.2 residue Trp-68 (Trp, data in E). The reopening rate is markedly reduced by substitution of cyclohexylalanine. G, magnified view of the M1–M2 helical interface where Trp-68 and Lys-170 come into close contact, illustrating the en face arrangement of these side chains.