Mutation of a single residue promotes gating of vertebrate and invertebrate two-pore domain potassium channels

Abstract

Mutations that modulate the activity of ion channels are essential tools to understand the biophysical determinants that control their gating. Here, we reveal the conserved role played by a single amino acid position (TM2.6) located in the second transmembrane domain of two-pore domain potassium (K2P) channels. Mutations of TM2.6 to aspartate or asparagine increase channel activity for all vertebrate K2P channels. Using two-electrode voltage-clamp and single-channel recording techniques, we find that mutation of TM2.6 promotes channel gating via the selectivity filter gate and increases single channel open probability. Furthermore, channel gating can be progressively tuned by using different amino acid substitutions. Finally, we show that the role of TM2.6 was conserved during evolution by rationally designing gain-of-function mutations in four Caenorhabditis elegans K2P channels using CRISPR/Cas9 gene editing. This study thus describes a simple and powerful strategy to systematically manipulate the activity of an entire family of potassium channels.

TOK channels use the two gates in classical K + channels to achieve outward rectification

“…The effects of the mutations we describe are consistent with studies of K 2P channels at the site equivalent to CaTOK-342 and ScTOK-316 that altered open probability and gating kinetics. 29,56 Studies of voltage-gated (1P) K + channels also align with this model; molecular dynamics simulations of Shaker-A471D (a site homologous to CaTOK-342 and ScTOK-G316), where replacement of the natural hydrophobic residue with a hydrophilic residue increased K + occupancy of the inner channel vestibule. 57 The observation that mutations inducing inward currents were only identified in S6 and not S8, likely reflects the asymmetry of the transmembrane domains that line the inner cavity of K 2P channels 25,32 and the evolutionary conservation of residues in S6 across TOK sequences, 58 suggestive of a prominent role in dictating gating and permeation characteristics.…”

“…CaTOK and ScTOK pore mutants show an increase in inward current when external RbCl replaces KCl, in line with previous observations that Rb + decreases the rate of pore-mediated C-type inactivation in KcsA, 62 Shaker 63 and Ca 2+ -activated K + channels 64 and induces stabilization of the open state of the selectivity filter gate in K 2P channels. 56 Furthermore, macromolecular crystallization studies of KcsA support the idea that Rb + ions stabilize its conductive conformation because the ions halt structural changes of the filter associated with C-type inactivation gating. 62 F I G U R E 8 Models illustrating proposed mechanism of TOK outward rectification and gating transitions.…”

Effects of the ventilatory stimulant, doxapram on human TASK‐3 (KCNK9, K2P9.1) channels and TASK‐1 (KCNK3, K2P3.1) channels

“…For L122D in particular, the mutation that caused the most dramatic reduction in doxapram effect, it has recently been shown that D or N mutations at this position in all K2P channels act as gain of function mutations 42 and mutations at this position alter the effectiveness of a number of K2P channel regulators. 42,43 This may confound interpretation of functional experiments that suggest this residue is involved in regulator binding. To address this, we investigated whether this particular mutation attenuated the effect of another known TASK-3 antagonist, zinc, which has been shown to act on residues on the extracellular side of the channel.…”

Clinical Importance of the Human Umbilical Artery Potassium Channels

“…The 2-pore domain K + (K 2P ) channels were first identified in the late 1990s [ 98 , 99 ]. This channel family produces leak-type K + currents [ 100 ] and are involved in controlling and stabilizing MP and the levels of cellular excitability [ 1 , 100 , 101 ].…”