Gábor Czirják, M.D., Ph.D.
Associate Professor, Department of Physiology, Semmelweis University
In the past two decades, I examined two-pore-domain (K2P) background potassium channels. The leak K+ current through these channels generally contributes to the stabilization of the resting membrane potential in several cell types, both in a wide variety of neurons and in the peripheral tissues. The activity of K2P channels is much less affected by the changes of the membrane potential than that of the voltage-gated or inwardly rectifying K+ channel types. However, K2P channels are regulated by extraordinary diverse stimuli, including mechano- and/or thermosensitivity, pH in the extra- or intracellular space or the composition of the lipid environment of the plasma membrane. Highly specific intracellular signaling mechanisms target and control these channels, and thereby adjust the excitability of different cell types under physiological and pathological conditions .
As I walked home slowly after work in the crowd of Örs Vezér square metro station, in a state of mind balanced at the border of imagination and reality, suddenly I had realized how I could examine the heterodimerization of TASK-1 and TASK-3 subunits. I plot ruthenium red sensitivity vs. pH sensitivity for the two parent homodimers and for the coexpressed subunits, and that may show whether heterodimers exist. Next day I began to design the experiments, and a few weeks later I had the data proving TASK-1/TASK-3 heterodimerization . It took a while to explain the data to the other members of the lab and make them accept the conclusion. Today we know that these heterodimers are major determinants of excitability of alpha motor neurons [3,4], cerebellar granule neurons , thalamocortical relay neurons , and the pH sensor glomus cells in the carotid body . It was more than ten years later that heterodimerization of other K2P channels could be demonstrated [8,9], and systematic analysis was performed by other laboratories, using previously unavailable technologies [10,11].
In the sequence of the human genome, being scarcely annotated at that time, I searched for novel potassium channel genes by a custom-designed computer program written in Delphi language. Because the computational power of my PC at the Department was quite limited, I installed the program on several PCs of the colleagues, and simultaneously searched the sequences of the different chromosomes, mostly overnight, for a week. This resulted in two previously unknown K+ channel genes (KCNV2 gene – Kv8.2 channel, and KCNK18 gene – TRESK channel, as we know them today). We finally succeeded in the cloning of these channels, using painstaking methods, which are considered as obsolete today [12,13]. I measured TRESK current first on the 21th of June in 2002 in the Xenopus oocyte heterologous expression system. I observed on the same day that TRESK is activated several-fold by the stimulation of endogenous Gq-coupled lysophosphatidic acid (LPA) receptors. Later I found that the stimulation of coexpressed Gq-coupled receptors, e.g. M1 muscarininc and AT1a angiotensin receptors also activate TRESK. This result was a key observation in my research carrier; since that time, my attention has been focused on TRESK channel. In the meanwhile, a Japanese group cloned human TRESK , so we have reported the mouse sequence and the unique regulation of the channel by Gq-coupled receptors and calcium for the first time .
TRESK channel has certainly been present since the origin of vertebrates, and it was possible to identify an ancient regulatory motif, a phosphorylation-dependent binding site for 14-3-3 adaptor protein, conserved throughout 400 million years of evolution . Even more interestingly, TRESK might have evolved further about 150 million years ago, during the early history of mammals, because a new set of regulatory elements emerged. These amino acid sequence motifs enable robust calcium-dependent activation of the channel, a unique mechanism of regulation within the K2P channel family. While the other background K+ channels are not affected by the elevation of cytoplasmic calcium concentration, TRESK is activated by dephosphorylation via the calcium/calmodulin-dependent phosphatase calcineurin . It was a great surprise and really rewarding in the pursuit to understand biological complexity that calcineurin turned out to directly bind to TRESK at a motif of the channel resembling the PxIxIT calcineurin-binding site of NFAT transcription factors . As an unexpected second turn, human TRESK was also shown to contain another binding site, analogous to the auxiliary LxVP calcineurin-binding motif of NFAT . This LQLP sequence in human TRESK fine-tunes the sensitivity of the channel to the calcium signal.
Between March of 2002 and 2010, I developed the project plans, designed the experiments and performed all the electrophysiological measurements in my first-author papers [12,13,15,16,18,19,20,21]. Before 2006, I wrote about 80 % of the manuscripts of these papers, whereas after 2006 more than 90 %. In 2010, our group was invited to write a review paper about K2P channels in the high impact Physiological Reviews . I have written the introduction chapter, perhaps my best-written text ever, and about half of the remaining part of the review. Afterwards, in several papers, I was corresponding author [17,22,23,24,25,26,27].
From the end of 2010 to January of 2016, I supervised a PhD student, Gabriella Braun [22,23,25,26]. She joined our laboratory during a successful period of research. For six years, the coexpression of more than 20 different serine/threonine kinase types with TRESK was tested in order to search for the one that phosphorylates the channel at a given important regulatory region and thus inhibits its K+ current. The ordinary guesses, such as protein kinase A and C or casein kinase II, all failed, and subsequently all the 15 other more exotic hypotheses too. Finally, a robust inhibition of TRESK by MARK2 kinase was demonstrated. Gabriella studied the interaction of MARK2 with TRESK . In another study, her original observation about the differential sensitivity of TREK-1 and TREK-2 channels to ruthenium red  helped the first identification of TREK-1/TREK-2 heterodimer by others and us [9,10]. After Gabriella defended her PhD thesis and went abroad, another PhD student, Enikő Pergel, joined our laboratory and began her work under my supervision. Enikő examined the mechanism of TRESK activation by novel-type protein kinase C , and defended her PhD thesis in February 2020.
As being an associate professor at the Department of Physiology of Semmelweis University, I also contribute to the teaching of medical students. According to the present official regulation, I am obliged to hold ten “contact” hours in a week (ten hours of seminars or lectures, which may also include four official hours of teaching a PhD student). About the life outside the Department, I am married and I am the father of three children.
- Enyedi P, Czirjak G. Molecular background of leak K+ currents: two-pore domain potassium channels. Physiol Rev. 2010; 90(2): 559-605.
- Czirjak G, Enyedi P. Formation of functional heterodimers between the TASK-1 and TASK-3 two-pore domain potassium channel subunits. J Biol Chem. 2002; 277(7): 5426-32.
- Berg AP, Talley EM, Manger JP, Bayliss DA. Motoneurons express heteromeric TWIK-related acid-sensitive K+ (TASK) channels containing TASK-1 (KCNK3) and TASK-3 (KCNK9) subunits. J Neurosci. 2004; 24(30): 6693-702.
- Larkman PM, Perkins EM. A TASK-like pH- and amine-sensitive ‘leak’ K+ conductance regulates neonatal rat facial motoneuron excitability in vitro. Eur J Neurosci. 2005; 21(3): 679-91.
- Kang D, Han J, Talley EM, Bayliss DA, Kim D. Functional expression of TASK-1/TASK-3 heteromers in cerebellar granule cells. J Physiol. 2004; 554(Pt 1): 64-77.
- Meuth SG, Aller MI, Munsch T, Schuhmacher T, Seidenbecher T, Meuth P, Kleinschnitz C, Pape HC, Wiendl H, Wisden W, Budde T. The contribution of TWIK-related acid-sensitive K+-containing channels to the function of dorsal lateral geniculate thalamocortical relay neurons. Mol Pharmacol. 2006; 69(4): 1468-76.
- Turner PJ, Buckler KJ. Oxygen and mitochondrial inhibitors modulate both monomeric and heteromeric TASK-1 and TASK-3 channels in mouse carotid body type-1 cells. J Physiol. 2013; 591(23): 5977-98.
- Blin S, Chatelain FC, Feliciangeli S, Kang D, Lesage F, Bichet D. Tandem pore domain halothane-inhibited K+ channel subunits THIK1 and THIK2 assemble and form active channels. J Biol Chem. 2014; 289(41): 28202-12.
- Lengyel M, Czirjak G, Enyedi P. Formation of Functional Heterodimers by TREK-1 and TREK-2 Two-pore Domain Potassium Channel Subunits. J Biol Chem. 2016; 291(26): 13649-61.
- Blin S, Ben Soussia I, Kim EJ, Brau F, Kang D, Lesage F, Bichet D. Mixing and matching TREK/TRAAK subunits generate heterodimeric K2P channels with unique properties. Proc Natl Acad Sci U S A. 2016; 113(15): 4200-5.
- Levitz J, Royal P, Comoglio Y, Wdziekonski B, Schaub S, Clemens DM, Isacoff EY, Sandoz G. Heterodimerization within the TREK channel subfamily produces a diverse family of highly regulated potassium channels. Proc Natl Acad Sci U S A. 2016; 113(15): 4194-9.
- Czirjak G, Toth ZE, Enyedi P. The two-pore domain K+ channel, TRESK, is activated by the cytoplasmic calcium signal through calcineurin. J Biol Chem. 2004; 279(18): 18550-8.
- Czirjak G, Toth ZE, Enyedi P. Characterization of the heteromeric potassium channel formed by kv2.1 and the retinal subunit kv8.2 in Xenopus oocytes. J Neurophysiol. 2007; 98(3): 1213-22.
- Sano Y, Inamura K, Miyake A, Mochizuki S, Kitada C, Yokoi H, Nozawa K, Okada H, Matsushime H, Furuichi K. A novel two-pore domain K+ channel, TRESK, is localized in the spinal cord. J Biol Chem. 2003; 278(30): 27406-12.
- Czirjak G, Vuity D, Enyedi P. Phosphorylation-dependent binding of 14-3-3 proteins controls TRESK regulation. J Biol Chem. 2008; 283(23): 15672-80.
- Czirjak G, Enyedi P. Targeting of calcineurin to an NFAT-like docking site is required for the calcium-dependent activation of the background K+ channel, TRESK. J Biol Chem. 2006; 281(21): 14677-82.
- Czirjak G, Enyedi P. The LQLP calcineurin docking site is a major determinant of the calcium-dependent activation of human TRESK background K+ channel. J Biol Chem. 2014; 289(43): 29506-18.
- Czirjak G, Enyedi P. TASK-3 dominates the background potassium conductance in rat adrenal glomerulosa cells. Mol Endocrinol. 2002; 16(3): 621-9.
- Czirjak G, Enyedi P. Ruthenium red inhibits TASK-3 potassium channel by interconnecting glutamate 70 of the two subunits. Mol Pharmacol. 2003; 63(3): 646-52.
- Czirjak G, Enyedi P. Zinc and mercuric ions distinguish TRESK from the other two-pore-domain K+ channels. Mol Pharmacol. 2006; 69(3): 1024-32.
- Czirjak G, Enyedi P. TRESK background K(+) channel is inhibited by phosphorylation via two distinct pathways. J Biol Chem. 2010; 285(19): 14549-57.
- Braun G, Nemcsics B, Enyedi P, Czirjak G. TRESK background K(+) channel is inhibited by PAR-1/MARK microtubule affinity-regulating kinases in Xenopus oocytes. PLoS One. 2011; 6(12): e28119.
- Enyedi P, Veres I, Braun G, Czirjak G. Tubulin binds to the cytoplasmic loop of TRESK background K(+) channel in vitro. PLoS One. 2014; 9(5): e97854.
- Czirjak G. PrinCCes: Continuity-based geometric decomposition and systematic visualization of the void repertoire of proteins. J Mol Graph Model. 2015; 62(118-27.
- Enyedi P, Braun G, Czirjak G. TRESK: the lone ranger of two-pore domain potassium channels. Mol Cell Endocrinol. 2012; 353(1-2): 75-81.
- Braun G, Lengyel M, Enyedi P, Czirjak G. Differential sensitivity of TREK-1, TREK-2 and TRAAK background potassium channels to the polycationic dye ruthenium red. Br J Pharmacol. 2015; 172(7): 1728-38.
- Pergel E, Lengyel M, Enyedi P, Czirjak G. TRESK (K2P18.1) Background Potassium Channel Is Activated by Novel-Type Protein Kinase C via Dephosphorylation. Mol Pharmacol. 2019; 95(6): 661-672.