Gábor Czirják, M.D., Ph.D.
Associate Professor, Department of Physiology, Semmelweis University
The main field of my scientific interest is the regulation of ion channels. In the past two decades, I mostly studied 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 changes of the membrane potential influence the activity of K2P channels much less than that of the voltage-gated or inwardly rectifying K+ channel types, however, they are regulated by an extraordinary diverse range of 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 .
Of course, only time will tell, but at present, it seems that my two best ideas in this field were the following:
As I slowly walked in the Örs Vezér square metro station with the crowd toward home, suddenly it came into my mind, how I can examine whether TASK-1 and TASK-3 K2P subunits form heterodimers. So far, it had been believed that K2P channels function as homodimers. I plot ruthenium red sensitivity vs. pH sensitivity for the two parent homodimers and for the coexpressed subunits and that may show if heterodimers exist. Next day I began the experiments, and next week 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 freshly published sequence of the human genome, which was poorly annotated at that time, I searched for novel potassium channel genes by my custom developed 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 examined the sequences of the different chromosomes simultaneously during the nights 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). Our laboratory finally succeeded in the cloning of these channels, although the cloning was more difficult at that time than today [12,13]. I measured TRESK current first on the 21th of June in 2002 and realized on the same day that TRESK is activated several-fold by the stimulation of endogenous Gq-coupled lysophosphatidic acid (LPA) receptors of the oocyte (later I demonstrated that the stimulation of other coexpressed Gq-coupled receptors, e.g. M1 muscarininc and AT1a angiotensin receptors also activate TRESK). This result was a key observation of my research carrier; since that time, my attention has been focused on TRESK channel. Unfortunately, we did not publish these data at once, mainly because they were considered uncertain, since TRESK was assembled from human genomic PCR fragments and not cloned from cDNA. Later our assembled sequence of human TRESK turned out to be identical to that published by a Japanese group . We have finally cloned TRESK from mouse cerebellar cDNA, and reported at least the mouse sequence and the unique regulation of the channel by Gq-coupled receptors and calcium for the first time .
I have been investigating the regulation of TRESK since that time. This channel has been present since the origin of vertebrates, and I identified an ancient regulatory motif, a phosphorylation-dependent binding site for 14-3-3 adaptor protein, which also controls channel activity, conserved throughout 400 million years of evolution . Even more interestingly, TRESK 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 soothing and rewarding feeling in the fever to understand biological complexity that I managed to demonstrate that calcineurin directly binds to TRESK at a motif of the channel resembling the PxIxIT calcineurin-binding site of NFAT transcription factors . As an unexpected second twist of the story, the analog of the auxiliary LxVP calcineurin-binding site of NFAT, could also be identified in human TRESK, and contributed to the regulation of the channel in response to the calcium signal .
Between March of 2002 and 2010, I developed the project plans, designed the experiments and performed all the electrophysiological measurements for the papers in which I was first author [12,13,15,16,18,19,20]. Before 2006, I wrote about 80 % of the manuscripts of these papers, whereas after 2006 more than 90 %. In 2010, our laboratory was invited to write a review paper about K2P channels in the highest impact Physiological Reviews . I have written the introduction chapter, which I consider one of my best-written texts ever, and about half of the remaining part of the review. I have also written the text of the responses to the Editor and Reviewers. At the same year, I concluded a study describing that two inhibitory kinase pathways converge on TRESK channel . I designed all the experiments, produced the constructs with the help of two enthusiastic technicians, performed all the electrophysiological measurements on my own, and wrote the manuscript. I managed the correspondence with the Reviewers at the Journal of Biological Chemistry about this paper, although I was not the official corresponding author. Afterwards I reported the results of my experiments as a corresponding author [17,22,23,24,25,26].
From the end of 2010 to January of 2016, I had the pleasure to direct the work of a PhD student, Gabriella Braun [22,23,25,26]. She joined our laboratory during a successful period of research. For six years, I have tested the coexpression of more than 20 different serine/threonine kinase types with TRESK 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 , and 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 lab and began her work under my supervision.
Although this text formed into an autobiography-like scientific introduction, I also paste here some general information about myself. 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 an assigned 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.