Editing of human KV1.1 channel mRNAs disrupts binding of the N-terminus tip at the intracellular cavity

Abstract

In the nervous system, A→I RNA editing has an important role in regulating neuronal excitability. Ligand-gated membrane receptors, synaptic proteins, as well as ion channels, are targets for recoding by RNA editing. Although scores of editing sites have been identified in the mammalian brain, little is known about the functional alterations that they cause, and even less about the mechanistic underpinnings of how they change protein function. We have previously shown that an RNA editing event (I400 V) alters the inner permeation pathway of human KV1.1, modifying the kinetics of fast inactivation. Here we show that the channel's inactivation gate enters deep into the ion permeation pathway and the very tip establishes a direct hydrophobic interaction with the edited position. By converting I to V, the intimacy of the interaction is reduced, allowing the inactivation gate to unbind with much faster kinetics.

A-to-I RNA Editing: Effects on Proteins Key to Neural Excitability

“…Miguel Holmgren and colleagues provided answers to these questions with exceptional clarity (Gonzalez et al, 2011). By substituting a cysteine at position 400, they were able to independently modify either the hydrophobicity or bulk at this site by direct chemical modification.…”

The emerging role of RNA editing in plasticity

“…Position 400 is at the apex of a region called the inner vestibule, immediately before the internal entrance of the ion conducting pore. The subtle change in hydrophobicity caused by editing hastens the dissociation of the inactivation particle from the inner vestibule (Gonzalez et al, 2011). Physiologically, this translates into a faster recovery from inactivation, an effect that would presumably enable higher action potential firing frequencies.…”

Regulation of Ion Channel and Transporter Function Through RNA Editing

“…The Shaker position corresponding to I400 was mutated to cysteine in order to serve as a target for chemical modification with methanethiosulphonate (MTS) reagents. As expected, the more hydrophobic a moiety attached to the edited codon, the slower the unbinding kinetics became (Gonzalez et al, 2011). In other words, once the inactivation particle is bound, hydrophobic interactions determine its off-rate.…”

“…We made six double cysteine channels with one cysteine at the edited position and the second between positions 2 and 7 at the N-terminus. Only with the construct 2C-470C did we observed an irreversible current reduction when the channels were exposed to an oxidizing environment, indicating that position 2 is the binding partner for the edited codon (Gonzalez et al, 2011). In summary, once valine substitutes for isoleucine, the intracellular cavity of K v 1.1 channels loses an important hydrophobic component to its association with the very tip of the N-terminus, an interaction that determines the off kinetics of the inactivation gate.…”