Modulation of Kv1.2 redox-sensitive gating by the transmembrane lectin LMAN2.

Abstract

The voltage-gated potassium channel Kv1.2 plays a pivotal role in the nervous system, where it regulates membrane excitability and action potential propagation. Compared to other Kv1 channels, Kv1.2 exhibits dynamic voltage-dependent gating with dramatic cell-to-cell variability that is attributed to regulation by unidentified external factors. Recent progress from our lab has identified extrinsic factors that shift the voltage-dependence of Kv1.2 gating. For example, the neutral amino acid transporter Slc7a5 causes a dramatic hyperpolarizing shift of Kv1.2 channel activation. A counteracting effect is that extracellular reducing conditions promote a depolarizing shift in channel activation. However, the underlying mechanism of redox sensitivity has been challenging to identify. Using a mass-spectrometry approach to identify novel Kv1.2 accessory proteins, followed by screening candidates with whole-cell patch clamp electrophysiology, we observed that co-expression with the transmembrane lectin LMAN2 caused a pronounced depolarizing shift of Kv1.2 voltage-dependence. Overexpression of LMAN2 enhanced Kv1.2 sensitivity to extracellular reducing conditions, while shRNA knockdown of endogenous LMAN2 abrogated the redox effects. Using a chimeric approach between Kv1.2 and the LMAN2 insensitive Kv1.5 subunit, we found the S2-S3 linker of the VSD to be essential for sensitivity to both LMAN2 and reducing conditions. Biotinylation experiments revealed that LMAN2 maturation to the plasma membrane is required for its effects on Kv1.2 gating. Furthermore, extracellular application of the membrane impermeable reducing agent TCEP to dorsal root ganglion (DRG) neurons, decelerated the activation kinetics of isolated Kv1.2 currents and increased excitability. In conclusion, we identified a novel accessory protein (the transmembrane Lectin, LMAN2) that alters Kv1.2 gating properties and contributes to redox sensitivity in heterologous cells and DRG neurons.