Peter Proks

Abstract:

Sleep apnea is a common disorder that represents a global public health burden. KCNK3 encodes TASK-1, a K⁺ channel implicated in the control of breathing, but its link with sleep apnea remains poorly understood. Here we describe a new developmental disorder with associated sleep apnea (developmental delay with sleep apnea, or DDSA) caused by rare de novo gain-of-function mutations in KCNK3. The mutations cluster around the ‘X-gate’, a gating motif that controls channel opening, and produce overactive channels that no longer respond to inhibition by G-protein-coupled receptor pathways. However, despite their defective X-gating, these mutant channels can still be inhibited by a range of known TASK channel inhibitors. These results not only highlight an important new role for TASK-1 K⁺ channels and their link with sleep apnea but also identify possible therapeutic strategies.

Abstract:

Oxytocin (Oxt) is known to regulate social communication, stress and body weight. The activation of Oxt receptors (OTR) has clinical potential to abate stress disorders and metabolic syndrome. Kamikihito (KKT) is a traditional Japanese medicine used to treat psychological stress-related disorders. We investigated the effects of KKT, its ingredients and chemical components on Oxt neurons and OTR. C-Fos expression was examined after oral and peripheral administration of KKT in rats. Electrophysiological change of Oxt neurons and Oxt release upon application of KKT were measured in rat brain slice. The direct effect of KKT, its ingredients and its chemical components were examined by cytosolic Ca²⁺([Ca²⁺]_i) measurement in Oxt neurons and OTR-expressing HEK293 cells. Both intraperitoneal and oral administration of KKT in rats induced c-Fos expression in neurons of the paraventricular nucleus (PVN) including Oxt neurons. Application of KKT induced activation of Oxt neurons and Oxt release. KKT increased [Ca²⁺]_i in OTR-expressing HEK293 cells, and failed to activate with OTR antagonist. KKT-induced PVN Oxt neuron activation was also attenuated by OTR antagonist. Seven chemical components (rutin, ursolic acid, (Z )-butylidenephtalide, p-cymene, senkunolide, [6]-shogaol, [8]-shogaol) of three ingredients (Zizyphi Fructus, Angelicae Acutilobae Radix, Zingiberis Rhizoma) from KKT had potential to activate OTR. KKT can directly activate PVN Oxt neurons by interacting with OTR. The interaction of seven chemical components from KKT may contribute to activate OTR. Effect of KKT on Oxt neurons and OTR may contribute to the treatment of Oxt related disorders.

Abstract:

In addition to the classical voltage-dependent behavior mediated by the voltage-sensing-domains (VSD) of ion channels, a growing number of voltage-dependent gating behaviors are being described in 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 mediated by the interaction of K+ with its selectivity filter. Recently, we 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 TREK-2 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 complex.

Abstract:

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.

Abstract:

The TREK subfamily of two-pore domain K+ (K2P) 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. However, 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 while remaining in the down conformation. We also show that the inhibitory effects of NFx are unrelated to its positive charge but can be influenced by agonists which alter filter stability, such as ML335, 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 also can 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 in which filter gating can be regulated to permit polymodal regulation.