Biology subjects

Proks, P.

Publications and source records attributed to Proks, P..

3 recordsLinked to original sources

bioRxiv2021

Effects of Ionic Strength on Gating and Permeation of TREK-2 K2P channels

In addition to the classical voltage-dependent behavior mediated by voltage-sensing-domains (VSD), a growing number of voltage-dependent gating behaviors are being described in ion channels that lack canonical VSDs. A common thread in their mechanism of action is the contribution of the permeating ion to this voltage sensing process. The polymodal K2P K+ channel TREK2 responds to membrane voltage through a gating process that is mediated by the interaction of K+ with its selectivity filter. Recently, we have found that this action can be modulated by small molecule agonists (e.g. BL1249) which appear to have an electrostatic influence on K+ binding within the inner cavity and produce an increase in the single-channel conductance of TREK-2 channels. Here, we directly probed this K+-dependent gating process by recording both macroscopic and single-channel currents of TREK-2 in the presence of high concentrations of internal K+. Surprisingly we found that the channel is inhibited by high internal K+ concentrations and that this is mediated by the concomitant increase in ionic-strength. However, we were still able to determine that the increase in single channel conductance in the presence of BL1249 was blunted in high ionic-strength, whilst its activatory effect (on channel open probability) persisted. These effects are consistent with an electrostatic mechanism of action of negatively charged activators such as BL1249 on permeation, but also suggest that their influence on channel gating is more complex.

biophysics↗

bioRxiv2020

A Mechanistic Basis for Inhibition of TREK-2 K2P Channels by Norfluoxetine

The TREK subfamily of Two-Pore Domain (K2P) K+ channels are inhibited by fluoxetine and its metabolite, norfluoxetine (NFx). Although not the principal targets of this antidepressant, TREK channel inhibition by NFx has provided important insights into the conformational changes associated with channel gating and highlighted the role of the selectivity filter in this process. But despite the availability of TREK-2 crystal structures with NFx bound, the precise mechanisms underlying NFx inhibition remain elusive. NFx has previously been proposed to be a state-dependent inhibitor, but its binding site suggests many possible ways in which this positively charged drug might inhibit channel activity. Here we show that NFx exerts multiple effects on single channel behavior that influence both the open and closed states of the channel, and that the channel can become highly activated by 2-APB whilst remaining in the down conformation. We also show that that the inhibitory effects of NFx are unrelated to its positive charge, but can be influenced by agonists such as ML335 which alter filter stability, as well as by an intrinsic voltage-dependent gating process within the filter. NFx therefore not only inhibits channel activity by altering the equilibrium between up and down conformations, but can also directly influence filter gating. These results provide further insight into the complex allosteric mechanisms that modulate filter-gating in TREK K2P channels and highlight the different ways that filter gating can be regulated to permit polymodal regulation.

biophysics↗

bioRxiv2020

Evaluating Inositol phospholipid interactions with Inward Rectifier Potassium Channels and characterising their role in Disease

Membrane proteins are frequently modulated by specific protein-lipid interactions. The activation of human inward rectifying potassium (hKir) channels by phosphoinositides (PI) has been well characterised. Here, we apply a coarse-grained molecular dynamics free-energy perturbation (CG-FEP) protocol to capture the energetics of binding of PI lipids to hKir channels. By using either a single- or multi-step approach, we establish a consistent value for the binding of PIP2 to hKir channels, relative to the binding of the bulk phosphatidylcholine phospholipid. Furthermore, by perturbing amino acid side chains on hKir6.2, we show that the neonatal diabetes mutation E179K increases PIP2 affinity, while the congenital hyperinsulinism mutation K67N results in a reduced affinity. We show good agreement with electrophysiological data where E179K exhibits a reduction in neomycin sensitivity, implying that PIP2 binds more tightly E179K channels. This illustrates the application of CG-FEP to compare affinities between lipid species, and for annotating amino acid residues.

biochemistry↗