Department of Physiology, Semmelweis University, Budapest
K2P leak K+ channels are major determinants of the resting membrane potential and regulate cellular excitability. The most recently discovered K2P subunit, TRESK is highly expressed in primary somatosensory neurons (including nociceptive afferents). TRESK is activated by the cytoplasmic Ca2+ signal, via the Ca2+/calmodulin-sensitive phosphatase calcineurin. Investigation of the physiological role of TRESK has been impeded by the lack of adequate modulators. We have recently reported that cloxyquin is a selective activator of TRESK.
In order to obtain TRESK modulators (activators with higher potency than cloxyquin or specific inhibitors), we created cloxyquin derivatives and tested their effect on TRESK and other K2P channels.
K2P channels were expressed in Xenopus oocytes and K+ currents were measured by two-electrode voltage clamp. Dorsal root ganglion (DRG) neurons were isolated from adult mice and K+ currents were measured by whole-cell patch clamp.
None of the synthesized 28 derivatives proved to be a more potent activator than cloxyquin. On the other hand several compounds were able to inhibit TRESK current. One of these derivatives (A2764) did not inhibit other K2P channels. IC50 of A2764 for TRESK was 24.2 µM. The degree of inhibition depended on the functional state of TRESK; if the channel was activated (by cytoplasmic Ca2+ signal), A2764 was more efficient (at 100 µM: 76.8±1.8% inhibition, n=6). In the resting state of TRESK the inhibition was smaller (45.4±4.3%, n=6). We tested the effect of A2764 on DRG cells; a subpopulation of the neurons was sensitive to A2764 (inhibition of the background K+ current was between 40 and 80%). This subpopulation was missing from TRESK-deficient DRG neurons.
A2764 is a selective inhibitor of TRESK and can be used to inhibit TRESK in native cells. This compound may be of use for modulating TRESK in animal models of nociceptive disorders.
Doctoral School: Doctoral School of Molecular Medicine
Doctoral Program: Cellular and Molecular Physiology
Supervisor: Péter Enyedi