January 1, 2014;
The unusual stoichiometry of ADP activation of the KATP channel.
KATP channels, oligomers of 4 pore-forming Kir6.2
proteins and 4 sulfonylurea receptors (SUR
), sense metabolism by monitoring both cytosolic ATP, which closes the channel by interacting with Kir6.2
, and ADP, which opens it via SUR
mutations that alter activation by ADP are a major cause of KATP channelopathies. We examined the mechanism of ADP activation by analysis of single-channel and macropatch recordings from Xenopus oocytes expressing various mixtures of wild-type SUR2A
and an ADP-activation-defective mutant. Evaluation of the data by a binomial distribution model suggests that wild-type and mutant SURs freely co-assemble and that channel activation results from interaction of ADP with only 2 of 4 SURs. This finding explains the heterozygous nature of most KATP channelopathies linked to mutations altering ADP activation. It also suggests that the channel deviates from circular symmetry and could function as a dimer-of-dimers.
Disease Ontology terms:
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Figure 1. Distinct responses of WT and mutant KATP channels to 300 μM ADP as an indicator of SUR-mediated ADP activation. (A) Concentration-dependent modulation by ADP of WT and mutant KATP channel currents recorded from inside-out patches. Numbers above bars indicate the number of patches included in the averages. (B–D) Representative recordings of macroscopic currents from inside-out patches excised from Xenopus oocytes co-injected with Kir6.2 and the indicated mixtures of WT and mutated (DNN) SUR2A.
Figure 2. All-or-none responses of single KATP channels to MgADP. (A,B) Recordings of single KATP channels in inside-out patches excised from oocytes injected with 0.02 ng of Kir6.2 RNA and 0.03 ng each of WT and mutant SUR2A RNAs. Compared to macroscopic current experiments, the amount of RNA injected was reduced 100-fold and smaller patch pipettes were used to lower the channel density toward 1 per patch. Amplitude histograms computed from the current records before (black) and ~10 s after (green) ADP application. (C) Using records as in (A) and (B), the ratios of the open probabilities (Po) measured after and before ADP application were calculated from amplitude histograms and revealed 2 clusters near 1 (as in A where ratio was 0.86) and near 0 (as in B where ratio was 0.03). The histogram plots average values of the ratio using either all values, or values above 0.5, or values below 0.5. (D) Mean effect of 300 μM ADP on macroscopic currents from WT, mutant, and an equal mix of WT and mutant channels. Numbers beside bars indicate the number of patches included in the averages.
Figure 3. Effect of ADP varies gradually with the ratio of WT to mutant SUR subunits. (A–H) MgADP responses of macroscopic currents recorded from Xenopus oocytes co-expressing Kir6.2 and the indicated mixtures of WT and mutant (DNN) SUR2A. (I) Average currents recorded in 300 μM ADP normalized to the current measured before in nucleotide-free solution. Numbers above bars indicate the number of patches included in the averages. Normalization was necessary because of the intrinsic variability of the oocyte system. The amplitudes in nA of the currents measured in 0 ATP before ADP application were on average (±s.e.m): 4.4 ± 0.6 (WT 1::0 DNN), 1 ± 0.5 (WT 0.8::0.2 DNN), 1.3 ± 0.4 (WT 0.6::0.4 DNN), 5.3 ± 1.4 (WT 0.5::0.5 DNN), 1.4 ± 0.4 (WT 0.4::0.6 DNN), 1.6 ± 0.7 (WT 0.2::0.8 DNN), 3.5 ± 1 (WT 0.16::0.84 DNN), and 1.5 ± 0.3(WT 0::1 DNN). This variability could not be attributed to a specific construct because experiments performed on the same day with the same batch of oocytes revealed no significant difference.
Figure 4. Model fitting reveals the stoichiometry of ADP activation of the KATP channel. (A) Assuming random assembly of WT and mutant subunits, the probability of occurrence of channels having exactly n WT subunits (Pn) follows a binomial distribution (equation shown at top). That probability Pn is shown as a function of the fraction of wild-type subunits (p) for each possible value of n as indicated above the curves. The label 2* corresponds to the probability of a channel having 2 adjacent (or 2 opposite) WT subunits. (B) Probability of a channel having n or more WT subunits calculated using the distributions of (A) (n indicated above the curves). The straight dotted line represents the probability of a WT channel if WT and mutant could not co-assemble. The symbols represent normalized ADP-activated current, calculated by normalization of the experimental data of Figure 3I. The rmsd of the experimental data from each model is: 0.327 (n = 4), 0.124 (n = 3), 0.119 (n = 2), 0.352 (n = 1), 0.031 (n = 2*), and 0.071 (no WT/mutant mixing; dotted straight line).