Golden Gate cloning enables efficient concatemer construction for biophysical analysis of heterozygous potassium channel variants from patients with epilepsy

Missense mutations that cause channelopathies usually occur in a heterozygous setting. Functional voltage-gated potassium channels are tetramers of pore-forming α-subunits. When a variant is co-expressed with the wild-type gene, six distinct tetramers can assemble. Conventional techniques cannot be used to study the tetramers individually because they rely on simultaneous expression of separate genes resulting in a mixture of all possible combinations. An established approach is generation of concatemeric constructs, but it is technically challenging and laborious for long cDNA. Here, we chose a missense variant of K_V1.6 channel with the V456L replacement found in patients with epilepsy. We applied Modular Cloning variation (MoClo) of the Golden Gate cloning technique to assemble six concatemeric constructs. We expressed all six channels in Xenopus laevis oocytes individually and investigated their biophysical properties by two-electrode voltage clamp. Incorporation of just one K_V1.6-V456L subunit was sufficient to shift the voltage dependence of deactivation, manifesting as a gain-of-function effect. Gating of concatemeric channels became increasingly affected as more mutant subunits were introduced. We modeled K_V1.6 in the open and closed states using AlphaFold 3 and available cryoEM structures of voltage-gated channels. The open state of the mutant channel was found overstabilized due to stronger contacts established by leucine compared to valine. Introduction of the measured properties of mutant K_V1.6 channels into a Hodgkin-Huxley-type model of an interneuron resulted in a dramatic increase of the excitation threshold. We conclude that concatemeric constructs are a facile instrument to investigate realistic heteromeric ion channels in health and disease.