Octameric stoichiometry of the KATP channel complex

Abstract

ATP-sensitive potassium (KATP) channels link cellular metabolism to electrical activity in nerve, muscle, and endocrine tissues. They are formed as a functional complex of two unrelated subunits-a member of the Kir inward rectifier potassium channel family, and a sulfonylurea receptor (SUR), a member of the ATP-binding cassette transporter family, which includes cystic fibrosis transmembrane conductance regulators and multidrug resistance protein, regulators of chloride channel activity. This recent discovery has brought together proteins from two very distinct superfamilies in a novel functional complex. The pancreatic KATP channel is probably formed specifically of Kir6.2 and SUR1 isoforms. The relationship between SUR1 and Kir6.2 must be determined to understand how SUR1 and Kir6.2 interact to form this unique channel. We have used mutant Kir6.2 subunits and dimeric (SUR1-Kir6.2) constructs to examine the functional stoichiometry of the KATP channel. The data indicate that the KATP channel pore is lined by four Kir6.2 subunits, and that each Kir6.2 subunit requires one SUR1 subunit to generate a functional channel in an octameric or tetradimeric structure.

Figure 1

K_ATP channels contain four Kir6.2 subunits. Macroscopic inside-out patch currents in response to voltage ramps (300 ms) between +100 mV (or c, +150 mV) and −100 mV, plotted versus membrane potential, for K_ATP channels formed from wild-type Kir6.2 (a, WT) or Kir6.2[N160D] (b, N160D) coexpressed with SUR1 subunits (in 1:1 molar ratio), or from mixed wild-type Kir6.2 and Kir6.2[N160D], plus SUR1 (c). (c, top) Currents are shown in the presence and absence of 20 μM spermine (spm), in each case after subtraction of leakage currents in the presence of 5 mM ATP (which reduces channel open probability to <1%). (c, bottom) Relative conductance (G_REL)-voltage relationships from the data shown above. The smooth lines in a (and b) are best fits of single Boltzmann functions to the data. For these patches, the unconstrained Boltzmann functions (for i = 5 and 0, respectively) were fitted with V ₅ = +220 mV, z ₅ = 0.4 (a); V ₁ = −54 mV, z ₁ = 2.9 (b). In c (bottom), the smooth line that superimposes on the data is the sum of five Boltzmann functions that correspond to different combinations of wild-type Kir6.2 and Kir6.2[N160D] in a tetramer, with fitted probability of wild-type incorporation (P) = 0.56. For this patch, the unconstrained Boltzmann functions (for i = 2–4) components, were fitted with V ₂ = −48 mV, z ₂ = 2.9; V ₃ = +9 mV, z ₃ = 2.9; V ₄ = +86 mV, z ₄ = 1.0. The individual Boltzmann functions are also plotted, with the contributing number of wild-type and Kir6.2[N160D] subunits indicated beside each curve. (d) Residual errors (calculated as the sum of squares of the difference between the fit and the data, every 1 mV, between −80 to +160 mV, excluding −10 to +10 mV), obtained by fitting with n = 2–6 subunits [Σ(G_REL − G_F)², where G_F is the fitted G_REL] plotted against the number of assumed subunits (N). The dashed line and open symbols correspond to the patch illustrated in c. Solid lines and symbols correspond to averaged data for n = 3–5 subunit fits for all patches (n = 9 patches, mean ± SEM). The trimeric model (n = 3) is inadequate to fit the data (see d), and better fits are not obtained by increasing n from 4 to 5. Similar results were obtained when attempting to fit the model to derivative (ΔG_REL/ΔV) data (not shown).

Figure 2

Fusion channels show identical voltage dependence to parental monomers. (a) Mean ± SEM for z _i of single Boltzmann functions fitted to G_REL-V relationships obtained from wild-type Kir6.2 or Kir6.2[N160D] subunits coexpressed with SUR1 (○), or from WTF or NDF fusion proteins (•), and for z _i of five component Boltzmann functions fitted to G_REL-V relationships obtained from mixed wild-type Kir6.2 and Kir6.2[N160D] subunits coexpressed with SUR1 (□), or from mixed expression of WTF and NDF fusion proteins (▪). For these mixed expressions, only z ₂, z ₃, and z ₄ were varied, z ₁ and z ₅ corresponded to the mean value fitted to homomeric expressions. The dashed line indicates the z _i values used to fit the tetrameric model to data in Fig. 6. (b) Mean ± SEM for V _i of single Boltzmann functions fitted to G_REL-V relationships obtained from wild-type Kir6.2 or Kir6.2[N160D] subunits coexpressed with SUR1 (○, n = 5 in each case), or from WTF or NDF fusion proteins (□, n = 4 in each case), and for V _i of five component Boltzmann functions fitted to G_REL-V relationships obtained from mixed wild-type Kir6.2 and Kir6.2[N160D] subunits coexpressed with SUR1 (•, n = 4), or from mixed expression of WTF and NDF fusion proteins (▪, n = 4). For these mixed expressions, only V ₂, V ₃, and V ₄ were varied, V ₁ and V ₅ corresponded to the mean value fitted to homomeric expressions. The dashed line indicates the V _i values used to fit the tetrameric model to data in Fig. 6.

Figure 6

A 1:1 SUR1/Kir6.2 stoichiometry is preferred. (a and b) Macroscopic inside-out patch currents in response to voltage ramps (300 ms) between +100 and −100 mV, plotted versus membrane potential, for K_ATP channels formed by coexpression of wild-type fusion proteins (WTF ) with Kir6.2[N160D] monomers (N160D) with (b) or without (a) SUR1 monomers (SUR). The approximate cDNA molar ratio of WTF/Kir6.2 [N160D]/SUR1 in transfection mixtures was 8:1:1. (top) Currents are shown in the presence and absence of 20 μM spermine (spm); in each case, currents are shown after subtraction of leakage currents in the presence of 10 mM ATP. (bottom) Relative conductance (G_REL)-voltage relationships from the data shown above. The superimposed continuous line is the sum of five Boltzmann functions with z _i and V _i parameters constrained as described in Fig. 2, a and b. In the fitting, only the apparent fraction of wild-type (P) and N160D mutant components (1 − P) was varied as indicated. (c and d) Macroscopic inside-out patch currents in response to voltage ramps (300 ms) between +100 and −100 mV, plotted versus membrane potential, for K_ATP channels formed by coexpression of fusion proteins (NDF ) with wild-type Kir6.2 monomers (WT) with (d) or without (c) SUR1 monomers (SUR). The approximate cDNA molar ratio of NDF/Kir6.2/SUR1 in transfection mixtures was 8:1:1. (top) Currents are shown in the presence and absence of 20 μM spermine (spm); in each case, currents are shown after subtraction of leakage currents in the presence of 5 mM ATP. (bottom) Relative conductance (G_REL)-voltage relationships from the data shown above. The superimposed continuous line is the sum of five Boltzmann functions with z _i and V _i parameters constrained as described in Fig. 3. For fitting, the apparent ratio of wild-type (P) and N160D mutant components (1 − P) was the only parameter varied as indicated.

Figure 3

Fusion channels contain four dimeric SUR1-Kir6.2 dimers. (a–c) (top) Macroscopic inside-out patch currents in response to voltage ramps (300 ms) between +100 and −100 mV, plotted versus membrane potential, for K_ATP channels formed from SUR1-Kir6.2 fusion proteins, containing either wild-type (a, WTF ) or N160D mutant (b, NDF ) Kir6.2 sequences, or from equimolar mixed WTF and NDF constructs (c). (top) Currents are shown in the presence and absence of 20 μM spermine (spm); in each case, currents are shown after subtraction of leakage currents in the presence of 10 mM ATP (which reduces channel open probability to <1%). (bottom) Relative conductance (G_REL)-voltage relationships from the data shown above. The continuous lines in b and c are best fits of single Boltzmann functions. For these patches, the unconstrained Boltzmann functions (for i = 5 and 0, respectively) were fitted with V ₅ = +200 mV, z ₅ = 0.4 (a); V ₁ = −58 mV, z ₁ = 2.9 (b). In c (bottom), the smooth line that superimposes on the data is the sum of five Boltzmann functions that correspond to different combinations of wild-type Kir6.2 and Kir6.2[N160D] in a tetramer, with fitted probability of wild-type incorporation (P) = 0.35. For this patch, the unconstrained Boltzmann functions (for i = 2–4), components were fitted with V ₂ = −46 mV, z ₂ = 2.9; V ₃ = +10 mV, z ₃ = 2.5; V ₄ = +99 mV, z ₄ = 1.2. The individual Boltzmann functions are also plotted, with the contributing number of wild-type and Kir6.2[N160D] subunits indicated beside each curve.

Figure 5

Monomeric Kir6.2 inhibits fusion protein conductance. ⁸⁶Rb⁺ efflux from untransfected COSm6 cells, and cells expressing SUR1-Kir6.2 fusion proteins with or without additional Kir6.2 and SUR1 subunits. Graphs show percent Rb⁺ released into the medium as a function of time in the presence of metabolic inhibitors (see methods) for a typical experiment (n = 3). The approximate cDNA molar ratio was 1:1:1 for SUR1-Kir6.2 fusion protein/Kir6.2/SUR1.

Figure 4

Fusion channels show reduced sensitivity to ATP. Representative currents recorded from inside-out membrane patches containing WTF or NDF K_ATP channels (as indicated) at −50 mV. Patches were exposed to differing [ATP] as indicated (mM). Inward currents are shown as upward deflections

Figure 7

Schematic representation of potential subunit arrangements in the K_ATP channel complex. The present results indicate that the K_ATP channel is an octamer consisting of Kir6.2 and SUR1 subunits in a 4:4 stoichiometry with Kir6.2 subunits forming the channel pore (i). The dominant negative effect of Kir6.2 on fusion protein expression suggests that stoichiometries other than 1:1 (between SUR1 and Kir6.2) form (ii) but do not generate functional channels, and that restitution of 1:1 stoichiometry by additional SUR1 subunits (iii) can regenerate functional complexes. However, since monomeric Kir6.2 subunits clearly can incorporate into functional channels with fusion proteins, albeit with low frequency, we suggest that the requisite SUR1 subunit can be provided, inefficiently, by a fusion protein whose Kir6.2 portion “hangs out” of the functional complex (iv).