Professor Stephen Tucker

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:

Pulmonary arterial hypertension (PAH) is a fatal disease of the cardiopulmonary system that lacks curative treatments. The main pathological event in PAH is elevated vascular resistance in the pulmonary circulation, caused by abnormal vasoconstriction and vascular remodelling. Ion channels are key determinants of vascular smooth muscle tone and homeostasis, and four PAH channelopathies (KCNK3, ABCC8, KCNA5, TRPC6) have been identified so far. However, the contribution of ion channels in other forms of PAH, which account for the majority of PAH patients, has been less well characterised. Here we reason that a variety of triggers of PAH (e.g. BMPR2 mutations, hypoxia, anorectic drugs) that impact channel function may contribute to the onset of the disease. We review the molecular mechanisms by which these 'extrinsic' factors converge on ion channels and provoke their dysregulation to promote the development of PAH. Ion channels of the pulmonary vasculature are therefore promising therapeutic targets because of the modulation they provide to both vasomotor tone and proliferation of arterial smooth muscle cells.

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 TWIK-related spinal cord potassium channel (TRESK) is encoded by KCNK18, and variants in this gene have previously been associated with susceptibility to familial migraine with aura (MIM #613656). A single amino acid substitution in the same protein, p.Trp101Arg, has also been associated with intellectual disability (ID), opening the possibility that variants in this gene might be involved in different disorders. Here, we report the identification of KCNK18 biallelic missense variants (p.Tyr163Asp and p.Ser252Leu) in a family characterized by three siblings affected by mild-to-moderate ID, autism spectrum disorder (ASD) and other neurodevelopment-related features. Functional characterization of the variants alone or in combination showed impaired channel activity. Interestingly, Ser252 is an important regulatory site of TRESK, suggesting that alteration of this residue could lead to additive downstream effects. The functional relevance of these mutations and the observed co-segregation in all the affected members of the family expand the clinical variability associated with altered TRESK function and provide further insight into the relationship between altered function of this ion channel and human disease.

Abstract:

The ability of spermatozoa to swim towards an oocyte and fertilize it depends on precise K+ permeability changes. Kir5.1 is an inwardly-rectifying potassium (Kir) channel with high sensitivity to intracellular H+ (pHi) and extracellular K+ concentration [K+]o, and hence provides a link between pHi and [K+]o changes and membrane potential. The intrinsic pHi sensitivity of Kir5.1 suggests a possible role for this channel in the pHi-dependent processes that take place during fertilization. However, despite the localization of Kir5.1 in murine spermatozoa, and its increased expression with age and sexual maturity, the role of the channel in sperm morphology, maturity, motility, and fertility is unknown. Here, we confirmed the presence of Kir5.1 in spermatozoa and showed strong expression of Kir4.1 channels in smooth muscle and epithelial cells lining the epididymal ducts. In contrast, Kir4.2 expression was not detected in testes. To examine the possible role of Kir5.1 in sperm physiology, we bred mice with a deletion of the Kcnj16 (Kir5.1) gene and observed that 20% of Kir5.1 knock-out male mice were infertile. Furthermore, 50% of knock-out mice older than 3 months were unable to breed. By contrast, 100% of wild-type (WT) mice were fertile. The genetic inactivation of Kcnj16 also resulted in smaller testes and a greater percentage of sperm with folded flagellum compared to WT littermates. Nevertheless, the abnormal sperm from mutant animals displayed increased progressive motility. Thus, ablation of the Kcnj16 gene identifies Kir5.1 channel as an important element contributing to testis development, sperm flagellar morphology, motility, and fertility. These findings are potentially relevant to the understanding of the complex pHi- and [K+]o-dependent interplay between different sperm ion channels, and provide insight into their role in fertilization and infertility.