Control of rectification and gating of cloned KATP channels by the Kir6.2 subunit

Abstract

KATP channels are a functional complex of sulphonylurea receptor (SUR1, SUR2) and inward rectifier K+ (Kir6.1, Kir6.2) channel subunits. We have studied the role of the putative pore forming subunit (Kir6.2) in regulation of rectification and gating of KATP channels generated by transfection of SUR1 and Kir6.2 cDNAs in COSm6 cells. In the absence of internal polyvalent cations, the current-voltage relationship is sigmoidal. Mg2+ or spermine4+ (spm) each induces a mild inward rectification. Mutation of the asparagine at position 160 in Kir6.2 to aspartate (N160D) or glutamate (N160E) increases the degree of rectification induced by Mg2+ or spermine4+, whereas wild-type rectification is still observed after mutation to other neutral residues (alanine-N160A, glutamine-N160Q). These results are consistent with this residue lining the pore of the channel and contributing to the binding of these cations, as demonstrated for the equivalent site in homomeric ROMK1 (Kir1.1) channels. Since Kir6.2 contains no consensus ATP binding site, whereas SUR1 does, inhibition by ATP has been assumed to depend on interactions with SUR1. However, we found that the [ATP] causing half-maximal inhibition of current (Ki) was affected by mutation of N160. Channels formed from N160D or N160Q mutant subunits had lower apparent sensitivity to ATP (Ki,N160D = 46.1 microM; Ki,N160Q = 62.9 microM) than wild-type, N160E, or N160A channels (Ki = 10.4, 17.7, 6.4 microM, respectively). This might suggest that ATP binding to the channel complex was altered, although examination of channel open probabilities indicates instead that the residue at position 160 alters the ATP-independent open probability, i.e., it controls the free energy of the open state, thereby affecting the "coupling" of ATP binding to channel inhibition. The results can be interpreted in terms of a kinetic scheme whereby the residue at Kir6.2 position 160 controls the rate constants governing transitions to and from the open state, without directly affecting ATP binding or unbinding transitions.

Figure 1

Single channel currents from K_ATP channels generated by expression of SUR1 with Kir6.2 subunits. (A) Representative single channel currents recorded from an inside-out patch containing wild-type K_ATP channel expressed from SUR1 and Kir6.2 subunits at the indicated voltages. (B) Current-voltage relationship for wild-type K_ATP channels in inside-out membrane patches, together with currents from K_ATP channels expressed from SUR1 and mutant Kir6.2 subunits as indicated. (C) Single channel currents recorded from an inside-out patch containing a K_ATP channel expressed from SUR1 and N160D mutant Kir6.2 subunits (top) or N160A mutant Kir6.2 subunits (bottom) at −50 mV. (D) Single channel currents recorded from an inside-out patch containing K_ATP channels expressed from SUR1 and N160Q mutant Kir6.2 subunits at −50 mV. 5 mM ATP was present for the time indicated by the horizontal bar. The fully closed state (c) and the fully open (75 pS, o) state are indicated. Movement artifacts obscure the record during the switch of the solution.

Figure 1

Single channel currents from K_ATP channels generated by expression of SUR1 with Kir6.2 subunits. (A) Representative single channel currents recorded from an inside-out patch containing wild-type K_ATP channel expressed from SUR1 and Kir6.2 subunits at the indicated voltages. (B) Current-voltage relationship for wild-type K_ATP channels in inside-out membrane patches, together with currents from K_ATP channels expressed from SUR1 and mutant Kir6.2 subunits as indicated. (C) Single channel currents recorded from an inside-out patch containing a K_ATP channel expressed from SUR1 and N160D mutant Kir6.2 subunits (top) or N160A mutant Kir6.2 subunits (bottom) at −50 mV. (D) Single channel currents recorded from an inside-out patch containing K_ATP channels expressed from SUR1 and N160Q mutant Kir6.2 subunits at −50 mV. 5 mM ATP was present for the time indicated by the horizontal bar. The fully closed state (c) and the fully open (75 pS, o) state are indicated. Movement artifacts obscure the record during the switch of the solution.

Figure 1

Single channel currents from K_ATP channels generated by expression of SUR1 with Kir6.2 subunits. (A) Representative single channel currents recorded from an inside-out patch containing wild-type K_ATP channel expressed from SUR1 and Kir6.2 subunits at the indicated voltages. (B) Current-voltage relationship for wild-type K_ATP channels in inside-out membrane patches, together with currents from K_ATP channels expressed from SUR1 and mutant Kir6.2 subunits as indicated. (C) Single channel currents recorded from an inside-out patch containing a K_ATP channel expressed from SUR1 and N160D mutant Kir6.2 subunits (top) or N160A mutant Kir6.2 subunits (bottom) at −50 mV. (D) Single channel currents recorded from an inside-out patch containing K_ATP channels expressed from SUR1 and N160Q mutant Kir6.2 subunits at −50 mV. 5 mM ATP was present for the time indicated by the horizontal bar. The fully closed state (c) and the fully open (75 pS, o) state are indicated. Movement artifacts obscure the record during the switch of the solution.

Figure 2

Substitution of negative charge at position 160 (D or E) induces strong inward rectification in the presence of spermine. (A) Representative currents recorded from inside-out membrane patches containing wild-type or mutant K_ATP channels, with mutations at position 160 in Kir6.2, as indicated. The membrane potential was stepped briefly from a holding potential of −20 to +100 mV and then to voltages between −80 and +10 mV (or +60 mV, N160Q). Currents were recorded in control (left), after inhibiting all K_ATP current with 5 mM [ATP] (middle), or 20 μM spermine (right). Residual linear conductance in 5 mM ATP is leak current in the patch. (B) Time constant of activation (τ_ACT, spermine unblock) versus membrane potential (mV) for representative currents recorded from inside-out patches containing N160D or N160E mutant channels in the presence of 20 μM spermine.

Figure 2

Substitution of negative charge at position 160 (D or E) induces strong inward rectification in the presence of spermine. (A) Representative currents recorded from inside-out membrane patches containing wild-type or mutant K_ATP channels, with mutations at position 160 in Kir6.2, as indicated. The membrane potential was stepped briefly from a holding potential of −20 to +100 mV and then to voltages between −80 and +10 mV (or +60 mV, N160Q). Currents were recorded in control (left), after inhibiting all K_ATP current with 5 mM [ATP] (middle), or 20 μM spermine (right). Residual linear conductance in 5 mM ATP is leak current in the patch. (B) Time constant of activation (τ_ACT, spermine unblock) versus membrane potential (mV) for representative currents recorded from inside-out patches containing N160D or N160E mutant channels in the presence of 20 μM spermine.

Figure 3

Mutation N160D in Kir6.2 induces strong rectification in the presence of Mg²⁺, as well as di- and trivalent polyamines. Representative currents recorded from inside-out membrane patches containing wild-type (WT, left) or N160D mutant (N160D, right) K_ATP channels in the absence (c) and presence of 1 mM Mg²⁺ (Mg), 100 μM putrescine (put), or 100 μM spermidine (spd). Quasi–steady-state current-voltage relationships were obtained by performing 1 s ramps from −100 to +100 mV.

Figure 4

Expression levels of N160 mutant channels. (A) ⁸⁶Rb⁺ efflux from untransfected COSm6 cells and cells expressing SUR1 and N160 mutant Kir6.2 subunits. Graphs show percent Rb⁺ released into the medium as a function of time in the presence of metabolic inhibitors for a typical experiment. (B) Estimated current density in patch-clamp experiments for wild-type and mutant channels. Bar graph shows mean current at −50 mV (in zero ATP and no added polyamines) ± SEM (n = 14–32 patches) measured over the first 5–10 s after isolation. Inactivation of N160Q channels during this time leads to underestimation of the peak N160Q current by ∼50% (*).

Figure 5

Mutations at position 160 in Kir6.2 alter [ATP] sensitivity of expressed channels. Representative currents recorded from inside-out membrane patches containing wild type or mutant K_ATP channels (as indicated) at −50 mV. Patches were exposed to differing [ATP] as indicated.

Figure 7

N160 mutations shift apparent ATP sensitivity and [ATP]-independent channel open probability. (A) Steady-state dependence of membrane current (relative to current in zero ATP) on [ATP] for wild-type and mutant K_ATP channels (as indicated). Data points represent the mean ± SEM (n = 3–8 patches). The fitted lines correspond to least-squares fits of the Hill equation (relative current = 100/ {1+([ATP]/K _i)^H}, with H = 1.8, and K _i = 10.4 μM (N160, wild type), 46.1 μM (N160D), 62.9 μM (N160Q), 17.7 μM (N160E), and 6.4 μM (N160A). (B) Relationship between estimated K _i and P_O(max) (see methods) for wild-type and mutant K_ATP channels. The curved line is a least squares fit of the empirical equation K _i,ATP = 263 · P_O(max) ¹¹.

Figure 6

Inactivation of N160Q mutant channels. (A) Representative currents recorded from inside-out membrane patches containing wild-type (A) or N160Q mutant (B) K_ATP channels (as indicated) at −50 mV. Patches were repeatedly exposed to 5 mM [ATP] as indicated by the bars above the records. In B, examples of both slowly inactivating (top) and rapidly inactivating (bottom) N160Q mutant channels are shown. (C) Time constant (τ) of inactivation of individual N160Q patches versus trial number after removal from 5 mM ATP. The time constant of inactivation does not change appreciably with time after patch excision within any given patch.

Figure 8

The gating effects of Kir6.2 N160 mutants can be explained by changes in the stability of the open state. (A) Model scheme for simulation of K_ATP channel activity. Only two sequential ATP binding steps are necessary to approximate the steepness of steady-state dependence of channel activity on [ATP] (see below). The open state is ATP independent (see text). (B) The steady-state [ATP] dependence of wild-type channels is well described by the model in A, with equilibrium constants as follows: K _A = 5 μM, K _CO = 5, K _OI = 0.1. The ATP dependence of N160D mutant channels is well described by the same model, but with K _CO = 80. (C) Model predicted K _i versus measured K _i (from the graphs in Fig. 7 A). Model predicted K _i is the [ATP] causing half-maximal inhibition of steady-state current for curves such as those shown in (B), with adjustments to the wild-type model as follows: wild type (WT), none; N160A, none; N160E, K _CO = 10; N160D, K _CO = 80; N160Q, K _CO = 80, K _OI = 1. (D) Modeled P_O(max) using adjustments above, versus measured P_O(max). Note that there is not agreement between measured and modelled P_O for N160Q mutant channels since the significant occupancy of the I state reduces the modelled P_O(max) to ∼0.5, although the slow transition rates between the I and O states would not be detectable by the method of experimental measurement of P_O (see text).

Figure 9

The model simulates the kinetic behavior observed in excised membrane patches. (A, top) The simulated kinetic scheme. (Bottom) Table showing the model parameters for individual rate constants indicated in the kinetic scheme. Only the rate constants boxed and bold were altered to produce the necessary simulations. (B) Simulated changes in P_O, in response to changes in [ATP].