Many voltage-gated potassium (Kv) channels display a time-dependent phenomenon called C-type inactivation, whereby prolonged activation by voltage leads to the inhibition of ionic conduction, a process that involves a conformational change at the selectivity filter toward a non-conductive state. Recently, a high-resolution structure of a strongly inactivated triple-mutant channel kv1.2-kv2.1-3m revealed a novel conformation of the selectivity filter that is dilated at its outer end, distinct from the well-characterized conductive state. While the experimental structure was interpreted as the elusive non-conductive state, our molecular dynamics simulations and electrophysiological measurements show that the dilated filter of kv1.2-kv2.1-3m is conductive and, as such, cannot completely account for the inactivation of the channel observed in the structural experiments. The simulation shows that an additional conformational change, implicating isoleucine residues at position 398 along the pore lining segment S6, is required to effectively block ion conduction. The I398 residues from the four subunits act as a state-dependent hydrophobic gate located immediately beneath the selectivity filter. By mutating I398 to Asparagine, ion permeation can be resumed in the kv1.2-kv2.1-3m channel, which was not a reversion C-type inactivation, since AgTxII fails to block the ionic permeation of kv1.2-kv2.1-3m_I398N. As a critical piece of the C-type inactivation machinery, this structural feature is the potential target of a broad class of QA blockers and negatively charged activators thus opening new research directions towards the development of drugs that specifically modulate gating-states of Kv channels.

Kainate receptors are key modulators of synaptic transmission and plasticity in the central nervous system. Different kainate receptor isoforms with distinct spatiotemporal expressions have been identified in the brain. The GluK1-1 splice variant receptors, which are abundant in the adult brain, have an extra fifteen amino acids inserted in the amino-terminal domain (ATD) of the receptor resulting from alternative splicing of exon 9. However, the functional implications of this post-transcriptional modification are not yet clear. We employed a multi-pronged approach using cryogenic electron microscopy, electrophysiology, and other biophysical and biochemical tools to understand the structural and functional impact of this splice insert in the extracellular domain of GluK1 receptors. Our study reveals that the splice insert alters the key gating properties of GluK1 receptors and their modulation by the cognate auxiliary Neuropilin and tolloid-like (Neto) proteins 1 and 2. Mutational analysis identified the role of crucial splice residues that influence receptor properties and their modulation. Furthermore, the cryoEM structure of the variant shows that the presence of exon 9 in GluK1 does not affect the receptor architecture or domain arrangement in the desensitized state. Our study thus provides the first detailed structural and functional characterization of GluK1-1a receptors, highlighting the role of the splice insert in modulating receptor properties and their modulation.