One-shot design elevates functional expression levels of a voltage-gated potassium channel

Membrane proteins play critical physiological roles as receptors, channels, pumps, and transporters. Despite their importance, however, low stability and expression levels often hamper their experimental characterization. We present an automated stability-design algorithm called mPROSS (https://mPROSS.weizmann.ac.il), which uses phylogenetic analysis and an atomistic potential, including an empirical lipophilicity scale. As a stringent test, we apply mPROSS to the Kv1.2-Kv2.1 paddle chimera voltage-gated potassium channel. Four designs, encoding 9-26 mutations relative to the parental channel, were functional and maintained potassium-selective permeation and voltage dependence in Xenopus oocytes with up to 14-fold increase in whole-cell current densities. Additionally, single-channel recordings reveal no change in the apparent channel-opening probability nor in unitary conductance, indicating that functional expression levels increase without impacting the activity profile of individual channels. These results suggest that other dynamical channels and receptors may be enhanced through one-shot design calculations.