TRP Pancreas

VLOGA IONSKIH KANALOV TRPM3 IN TRPM5 PRI URAVNAVANJU MREŽNE AKTIVNOSTI V OTOČKIH TREBUŠNE SLINAVKE

Osnovni podatki o projektu

Evidenčna številka projekta: N3-0048 (D)

Vodja projekta: prof. dr. Marjan Slak Rupnik (1.7.2016-31.12.2017)

                    doc. dr. Andraž Stožer (od 1.1.2018)

Trajanje projekta: 1.7.2016 - 30.06.2020

Obseg v letih 2017 in 2018: 1,01 FTE

Klasifikacija ARRS:

Koda: 3.07.00

Veda: Medicina      

Področje: Metabolne in hormonske motnje

POVZETEK VSEBINE PROJEKTA:

Znano je, da so K_ATP kanali glavni ionski kanali, ki regulirajo izločanje inzulina iz trebušne slinavke. Privzem glukoze in njen metabolizma v celicah beta povzroči zapiranje K_ATP kanalov, depolarizacijo celic beta, vtok kalcijevih ionov in s kalcijem sproženo zlivanje inzulinskih veziklov s plazmalemo celic beta. Vendar pa zadnja leta pa postaja znano, da imajo pomembno vlogo pri uravnavanju izločanja inzulina tudi drugi ionski kanali, še posebno t.i. TRP (angl. transient receptor potential) ionski kanali, ki jih celice beta bogato izražajo. Kljub naraščajočem številu raziskav, ki preučujejo izražanje kanalov TRP v celicah beta, še vedno ne vemo, kako in pod katerimi pogoji kanali TRP aktivirajo celice beta ali kako vplivajo na električno aktivnost, kalcijevo signalizacijo ali izločanje inzulina. Z uporabo elektrofizioloških pristopov, meritve kalcijeve dinamike s pomočjo konfokalne mikroskopije in naprednih metod mrežne analize raziskujemo specifično vlogo dveh izmed kanalov TRP, to je TRPM3 in TRPM5. Delovna hipoteza, ki jo preverjamo s pomočjo tega projekta, je, da sta TRPM3 in TRPM5 pomembno vključena v sprožitev ali vzdrževanje mrežne aktivnosti celic beta, ki jo povzroči draženje z glukozo, s tem pa vplivata na dinamiko sproščanja inzulina v normalnih in patoloških pogojih. Boljše razumevanje celične regulacije in funkcije teh dveh kanalov bo omogočilo nov vpogled v regulacijo izločanja inzulina pri zdravih ljudeh in diabetičnih bolnikih. Ker so v nekaterih primerih znane molekularne tarče in učinek kanalov TRP (TRPM3 za regulacijo bolečine in TRPM5 za modulacijo okusa), bi razjasnitev vloge kanalov TRP v celicah beta omogočila razvoj novih zdravil za zdravljenje v humani medicini.

 PROJECT OUTLINE

Network activity in pancreatic islets

Islets of Langerhans in the pancreas supply insulin to the organism in response to changing metabolic demand. Lack of insulin function, due to impairment in insulin release or insulin-induced signaling results in diabetes mellitus, a public health threat that has become one of the most prevailing human illnesses world-wide [1].

Islets of Langerhans consist of different cell types, the α, b, g, d, and e cells, which secrete distinct hormones directly into the bloodstream. The b cells, secreting insulin, form by far the largest group. Recent technological advancements have enabled functional multicellular calcium imaging (fMCI) to assess cellular activity with unprecedented spatial and temporal resolution and to support the analysis of the nutrient-induced signaling in intact pancreatic islets using advanced analytical tools from the realm of complex network theory (Fig. 1) [2] . This revealed that beta cells form a broad-scale small-world network with clear functional sub-compartments (communities) with correlated electrical activity, calcium signaling and calcium-dependent insulin release [3, 4]. The higher the glucose concentration, the more communities become active and the stronger the interconnectivity between cells communities (Fig. 1) [5]. The correlated broad-scale topology of network activity implies particular dynamic properties regarding stability, rapid information propagation speed, and a high degree of synchronizability. Most importantly, all these features cooperate to optimize insulin release [3]. The mechanisms that mediate/modulate network activity in pancreatic islets are poorly understood.

TRP channels in pancreatic β-cells

It is textbook knowledge that ATP-sensitive K⁺ (K_ATP) channels act as the master regulators of insulin release [6] . Closure of K_ATP channels following glucose uptake and metabolism causes membrane depolarization in beta cells, which then triggers Ca²⁺ influx and Ca²⁺-dependent insulin exocytosis. Sulfonylureas that inhibit K_ATP are widely used to enhance insulin release in patients with diabetes type 2 and in some forms of neonatal diabetes [7] .

However, in recent years, it is becoming increasingly clear that the repertoire of ion channels in pancreatic β-cells is much larger than previously believed. In particular, several members of the Transient Receptor Potential (TRP) superfamily were shown to be expressed at different levels in β-cells (Fig. 2) [8].

TRP channels form a class of cation-permeable channels that can be seen as dedicated cellular sensors, responding to a surprising variety of exogenous and endogenous chemical and physical stimuli [9]. The 28 mammalian members of the TRP superfamily have been implicated in a variety of physiological processes, including somatosensation and pain, Ca²⁺ and Mg²⁺ (re)absorption, cardiovascular function, taste perception and more. TRP channels represent appealing and heavily pursued new targets for the treatment of human diseases [10].

Despite the growing number of studies describing TRP channel expression in pancreatic β-cells, it is largely unclear how and under which conditions these channels are activated/regulated in these cells, or how their activity affects the electrical activity, calcium signalling and insulin release in the context of the intact pancreas [11]. In this project, we propose to combine single-cell electrophysiological assays with fMCI-supported network analyses in pancreatic slices to investigate the specific role of two TRP channels, TRPM3 and TRPM5.

TRPM3

TRPM3, a calcium-permeable nonselective cation channel, represents a typical example of a polymodally gated TRP channel, in that it can be activated by ligands such as the neurosteroid pregnenolone sulphate (PS), by heat, and by membrane depolarization [12-14]. A unique feature of TRPM3 is that it actually contains two distinct pathways for ions: under basal conditions or upon stimulation with heat or PS, ion permeation occurs through the central pore, which is relatively selective for Ca²⁺ ions [15]. However, co-stimulation with drugs, such as clotrimazole, tamoxifen and structurally related compounds can also lead to opening of an alternative permeation pathway, which allows massive influx of Na⁺ ions [15].

TRPM3 is highly expressed in somatosensory neurons, where it plays a decisive role in the nocifensive response to PS and heat, and in the development of heat hyperalgesia during inflammation [13]. Therefore, TRPM3 forms an attractive target for potential novel analgesics [16]. In addition, functional TRPM3 is also found in pancreatic β-cells, but its physiological role in these cells is unclear [14, 17]. We recently described that a novel potent synthetic TRPM3 agonist, CIM0216, can evoke insulin release from intact islets [18]. However, the signals that activate TRPM3 in islets under normal or pathophysiological conditions are currently unclear. To date, its potential contribution to initiating or sustaining network activity has not been studied

TRPM5

TRPM5 is a calcium-activated but calcium-impermeable cation channel that is best known for its role as transducer channel in taste receptor cells for sweet, bitter, and umami [19]. Interestingly, apart from its essential role in transducing the sweetness of glucose in the tongue, TRPM5 also appears to modulate the response of pancreatic islets to a rise in plasma glucose, and thereby regulates insulin secretion [20, 21]. In particular, it was shown that TRPM5-deficient mice show impaired glucose tolerance, which was associated with a loss of high-frequency calcium oscillations in intact islets [20]. Oppositely, it was found that TRPM5 expression is strongly down-regulated in mouse models of diabetes, which may contribute to impaired insulin signalling in these animals [22]. Moreover, it was also shown that TRPM5 is required for the fructose-induced potentiation of glucose-induced islet activity and insulin release, downstream of activation of sweet taste receptors [23]. Finally, genetic variation within the TRPM5 locus was shown to be associated with pre-diabetic phenotypes in humans [24]. Thus, TRPM5 represents a potentially interesting target for the normalisation of glucose homeostasis in diabetes mellitus. Indeed, our unpublished results indicate that a TRPM5 agonist improves glucose tolerance in a preclinical model of diet-induced diabetes. However, at this point, it is largely unclear how TRPM5 influences the electrical signalling in individual beta cells and network activity in intact islets.

The overall goal of this project is to obtain important new insight into the role of two members of the large TRP superfamily, namely TRPM3 and TRPM5, in glucose-induced signalling in intact pancreatic islets. Based on our published and unpublished data, we will investigate the hypothesis that these two cation channels can have a profound influence on the initiation or maintenance of glucose-induced β-cell network activity, and thus shape insulin release under normal and pathological conditions. A better understanding of the cellular regulation and functional impact of these two channels will not only provide new insights into the regulation of insulin release, but may also provide new avenues for controlling glucose homeostasis in diabetes. Moreover, since small-molecule compounds targeting these channels are currently being tested in preclinical trials for the treatment of pain (TRPM3) or the modulation of taste (TRPM5), knowledge of the role of these channels in islet function is crucial for the further development of such compounds towards use in humans.

Scientific aims of the project will be addressed in six work packages (WPs):

Aim 1: Regulation of TRPM3 activity by cytosolic ATP and PIP₃ (Performed by international collaborator from KU Leuven, Belgium)

Recent unpublished data indicate that TRPM3 activity is inhibited by cytosolic ATP and strongly potentiated by the membrane phospholipid phosphatidylinositol 3,4,5-bisphosphate (PIP₃). These cellular regulation mechanisms are potentially highly relevant in the context of pancreatic β-cell, where intracellular ATP levels are directly dependent on plasma glucose levels, whereas PIP₃ levels can rapidly rise upon stimulation of insulin receptors (autocrine) or of other tyrosine kinase receptors such those for glucagon-like peptide 1 (GLP-1) or insulin-like growth factor (IGF-1).

Collaboration group from Leuven will perform detailed biophysical experiments and structure-function analyses to determine the mechanisms and protein domains in TRPM3 that underlie regulation by ATP and PIP₃ (WP1: Mechanistic insight into the regulation of TRPM3 activity by cytosolic ATP and PIP₃), and assess how these fundamental cellular signals influence TRPM3 activity during glucose stimulation in pancreatic β-cells (WP2: WP2: ATP- and PIP₃-dependent regulation of TRPM3 in b cells).

Aim 2: Impact of TRPM3 and TRPM5 on glucose-induced network activity in intact islets (Performed by research group from Institute of Physiology, Faculty of Medicine, Slovenia)

In recent years, research group from KU Leuven has acquired important expertise and unique experimental tools to address the function of TRPM3 and TRPM5 in cells, organs and intact organisms. These include TRPM3^-/- and TRPM5^/- mouse lines, as well as specific agonists and antagonists for both channel types. Our research group from will apply these tools to directly and selectively evaluate the role of stimulation/inhibition of TRPM3 and TRPM5 on β cell network activity in intact pancreatic islets. This corresponds to WP3: Modulation of network activity in intact islets by TRPM3 and WP4; Modulation of network activity in intact islets by TRPM5.

Aim 3: TRPM3 and TRPM5 function in diabetic models (Performed by international collaborator from KU Leuven, Belgium and by research group from Institute of Physiology, Faculty of Medicine, Slovenia)

Electrical activity, expression of ion channels and β cell network parameters in pancreatic islets can change significantly in response to diabetes. Both collaborators will analyse to what extent network activity and functional expression of TRPM3 and TRPM5 is affected in animal models of diabetes. Moreover, we will assess whether (ant)agonists of these channels can be used to normalize β cell network activity under these conditions (WP5: Altered TRPM3 and TRPM5 function and network activity in diabetic models).

Aim 4: Establishment of a mathematical model of network activity (Performed by research group from Institute of Physiology, Faculty of Medicine, Slovenia).

Based on the results obtained in WP1-5, we will develop a predictive mathematical model describing the activity of β-cell network, including the contribution of TRPM3 and TRPM5, and the alterations in diabetic animals before and after pharmacological modulation of the activity of these channels (WP6: Development of a mathematical model of network activity).

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3. Stozer, A., Gosak, M., Dolensek, J., Perc, M., Marhl, M., Rupnik, M.S., and Korosak, D. (2013). PLoS Comput Biol 9, e1002923.
4. Hodson, D.J., and Mollard, P. (2013). J Neuroendocrinol 25, 674-675.
5. Markovic, R., Stozer, A., Gosak, M., Dolensek, J., Marhl, M., and Rupnik, M.S. (2015). Sci Rep 5, 7845.
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18. Held, K., Kichko, T., De Clercq, K., Klaassen, H., Van Bree, R., Vanherck, J.C., Marchand, A., Reeh, P.W., Chaltin, P., Voets, T., et al. (2015). Proc Natl Acad Sci U S A.
19. Zhang, Y., Hoon, M.A., Chandrashekar, J., Mueller, K.L., Cook, B., Wu, D., Zuker, C.S., and Ryba, N.J. (2003). Cell 112, 293-301.
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RAZISKOVALCI

Dr.       Dolenšek Jurij                          SICRIS

Dr.       Duh Andrej                              SICRIS

Farasin Darja                                      SICRIS

Dr.       Gosak Marko                           SICRIS

Dr.       Korošak Dean                          SICRIS

Dr.       Križančić Bombek Lidija          SICRIS

           Lukaček Zvezdana                   SICRIS

Dr.       Majdič Gregor                        SICRIS

Dr.       Marhl Marko                           SICRIS

           Mlakar Rudi                             SICRIS

            Pohorec Viljem                       SICRIS

Dr.       Skelin Klemen Maša              SICRIS

Dr.       Slak Rupnik Marjan                SICRIS

Dr.       Stožer Andraž                         SICRIS

Vodopivc Peter                                   SICRIS

Bibliografske reference, ki izhajajo iz projekta

GOSAK, Marko, STOŽER, Andraž, MARKOVIČ, Rene, DOLENŠEK, Jurij, PERC, Matjaž, RUPNIK, Marjan, MARHL, Marko. Critical and supercritical spatiotemporal calcium dynamics in beta cells. Frontiers in physiology, ISSN 1664-042X, 2017, vol. 8, str. 1-17, ilustr., doi: 10.3389/fphys.2017.01106. [COBISS.SI-ID 512760376]

SKELIN, Maša, DOLENŠEK, Jurij, RUPNIK, Marjan, STOŽER, Andraž. The triggering pathway to insulin secretion : functional similarities and differences between the human and the mouse [beta] cells and their translational relevance. Islets, ISSN 1938-2022, ilustr. http://www.tandfonline.com/doi/full/10.1080/19382014.2017.1342022, doi: 10.1080/19382014.2017.1342022. [COBISS.SI-ID 512726328]

DOLENŠEK, Jurij, POHOREC, Viljem, RUPNIK, Marjan, STOŽER, Andraž. Pancreas physiology. V: SEICEAN, Andrada (ur.). Challenges in pancreatic pathology. Rijeka: InTech. cop. 2017, str. [19]-52, ilustr. https://cdn.intechopen.com/pdfs-wm/53020.pdf, doi: 10.5772/65895. [COBISS.SI-ID 512723000]

GOSAK, Marko, MARKOVIČ, Rene, DOLENŠEK, Jurij, RUPNIK, Marjan, MARHL, Marko, STOŽER, Andraž, PERC, Matjaž. Network science of biological systems at different scales : a review. Physics of life reviews, ISSN 1873-1457. http://www.sciencedirect.com/science/article/pii/S1571064517301501, doi: 10.1016/j.plrev.2017.11.003. [COBISS.SI-ID 512746040]

GOSAK, Marko, MARKOVIČ, Rene, DOLENŠEK, Jurij, RUPNIK, Marjan, MARHL, Marko, STOŽER, Andraž, PERC, Matjaž. Loosening the shackles of scientific disciplines with network science : reply to comments on "Network science of biological systems at different scales: a review". Physics of life reviews, ISSN 1571-0645, 2018, vol. 24, str. 162-167, doi: 10.1016/j.plrev.2018.01.008. [COBISS.SI-ID 23696648]

KOROŠAK, Dean, RUPNIK, Marjan. Collective sensing of ß-cells generates the metabolic code. Frontiers in physiology, ISSN 1664-042X, Jan. 2018, vol. 9, article 31, str. 1-8, ilustr. https://dk.um.si/IzpisGradiva.php?id=69470, doi: 10.3389/fphys.2018.00031. [COBISS.SI-ID 21119766]c  

REALIZACIJA PROJEKTA marec 2018

Cilj 1: Regulacija aktivnosti TRPM3 s citosolnim ATP in PIP3.

Elektrofiziološke meritve (različne tehnike vpete krpice membrane) izvaja skupina I, ki odda svoje poročilo.

Cilj 2: Vpliv TRPM3 in TRPM5 na mrežno aktivnost, povzročeno z glukozo, v normalnih otočkih.

[1] V preglednem članku smo opisali sprožitveni mehanizem za izločanje inzulina pri miškah in ljudeh. Prenos glukoze v celice beta, njihov metabolizem v mitohondrijih in citoplazmi, nastanek ATP, zmanjšanje prevodnosti od ATP odvisnih K kanalov (KATP), depolarizacija plazmaleme in povečan vnos kalcija v celice skozi napetostno odvisne kalcijeve kanale sproži zlivanje veziklov z inzulinom. TRP kanali imajo zelo verjetno vlogo pri depolarizacijskih Na ali Ca tokovih, ki membrano celic beta depolarizirajo po tem, ko se zaprejo KATP kanali. Raziskave pri miškah zaenkrat potrjujejo vlogo TRPM5 pri tem procesu, saj imajo miške z izbitim genom za TRPM5 zmanjšan od kalcija odvisen kationski tok [1]. Delo je v tekočem letu že prejelo dva citata (vir Google Scholar).

[2] V tem poglavju v učbeniku smo natančno in pregledno opisali fiziologijo trebušne slinavke, predvsem pri ljudeh. Za razumevanje funkcije trebušne slinavke je nenadomestljivo znanje o makro- in mikro-strukturi žleze, o njenem ožiljenju in oživčenju, ki predstavlja osnovo za razumevanje razvoja bolezni [2]. Delo je do sedaj že prejelo en citat (vir Google Scholar).

[3] COBISS.SI-ID 512760376 Koordinirano delovanje celic v Langerhansovem otočku je posledica oscilatornih sprememb membranskega potenciala in kalcijeve dinamike. Presledkovni stiki omogočajo prenos informacije med celicami beta, ki v otočku tvorijo kompleksen sincicij.  V prvih nekaj minutah po stimulaciji z glukozo verjetnostno funkcijo velikosti kalcijevih valov opiše potenčni odnos, kar opišemo kot kritično obnašanje sistema. Po tem obdobju se delež velikih valov poveča do te mere, da sistem postane superkritičen. In vivo pogoje boljše opiše oscilatorna stimulacija, kjer smo zasledili le kritično obnašanje sistema, tako v eksperimentalnih podatkih kot v matematičnem modelu. Vloga kanalov TRP pri prehodu sistema iz kritičnega v superkritičnega in vloga kanalov TRP pri ohranjanju kritičnega sistema med oscilatorno stimulacijo še ni znana [3]. [2]. Delo je do sedaj že prejelo dva citata (vir Google Scholar).

[4, 5] COBISS.SI-ID 512746040 Vpeljava novih metod s področja večplastnih mrež za analizo kolektivne dinamike pankreasnih celic beta. Tovrstni pristopi predstavljajo novo vejo v domeni mrežnih znanosti in so primerni predvsem za opis kompleksnih mrež, ki se spreminjajo s časom ter za sisteme, kjer imamo več vrst interakcij in kjer je dinamika multimodalna. Vse od naštetega je značilno tudi za sincicij celic beta in omogoča drugačen pristop k določitvi vloge kanalov TRP v fiziologiji celice beta. Novo metodologijo smo podrobno opisali in demonstrirali za primer sočasnih meritev kalcija, membranskega potenciala in sekrecije inzulina v celicah beta ter za primer preučevanja vpliva farmakoloških substanc. Članek je bil objavljen v visoko uvrščeni reviji Physics of Life Reviews (IF 13) [4, 5]. Poleg tega so bili rezultati v obliki predavanj predstavljeni na dveh konferencah (dr. Gosak, dr. Stožer). Delo je do sedaj že prejelo en citat (vir Google Scholar).

[6] COBISS.SI-ID 21119766 V tem članku opišemo inovativen model za opis kolektivne dinamike celic beta, ki temelji na t.i. spin glass modelu. Analizirali smo skupino Ca2+ signalov izmerjenih v akutni rezini tkiva trebušne slinavke v fizioloških pogojih. Ugotovili smo obstoj močno koreliranih stanj, ki sobivajo s pretežno šibkimi parskimi korelacijami razširjenimi po celotnem otočku. Kolektivna aktivnost znotrajceličnega Ca2+ v otočku kaže intermitentne lastnosti, skaliranje amplitude konic signalov in avto-asociativne spominske lastnosti v odvisnosti od stimulusa. Za opis funkcionalne mreže kolektiva celic beta smo uporabili preprost model spinskega stekla, ki uspešno opiše te ugotovitve. Pokazali smo, da so znotrajcelični Ca2+ signali, proizvedeni s kolektivnim zaznavanjem celic beta, sestavni del metabolne kode, ki uravnava sproščanje insulina in omejuje velikost otočka. Vloga TRP kanalov pri kolektivni dinamiki še ni znana. Delo je do sedaj že prejelo en citat (vir Google Scholar).

Nastale težave in pristop k reševanju

Skupina je razvila izredno močno orodje za snemanje kalcijeve dinamike, to je kombinacijo tkivne rezine trebušne slinavke in konfokalne mikroskopije. Pri raziskovanju odzivov mišk, z izbitim genom za TRPM3 in TRPM5 smo naleteli na nepričakovane težavo. Po draženju z fiziološkimi in nadfiziološkimi koncentracijami glukoze se celice beta teh mišk odzovejo drugače kot pri kontrolnih miškah. V kontrolnih miškah se večina celic beta odzove zelo podobno, a zasledimo razlike med otočki, pri miškah z izbitim genom za TRPM3 in TRPM5 pa smo opazili zelo različne tipe odzivov celic že znotraj posameznih otočkov. To predstavlja veliko težavo za detekcijo kalcijeve dinamike, saj je potrebno tipe odzivov v prvem koraku določiti in v naslednjem koraku prilagoditi parametre za analizo za vsak tip odzivov posebej. Analiz nestandardnih in kvalitativno različnih tipov kalcijevih odzivov tudi znotraj enega otočka smo se lotili na dva načina: i) z algoritmom za hierarhično grupiranje, s katerim želimo robustno določiti število in vrsto dinamičnih fenotipov v posnetkih posameznega otočka. Grupiranje je osnovano na podlagi korelacij med dinamičnimi odzivi in na osnovi dinamičnih lastnosti časovnih vrst, predvsem na Fourierjevih frekvenčnih spektrih. ii) Z izdelavo novega in inovativnega algoritma, ki omogoča kvantifikacijo časovnih vrst izven okvirjev standardnih pristopov (določevanje frekvenc in trajanj oscilacij ter koaktivnosti celic), ki se uporabljajo za analize normalnih odzivov. Osnovna ideja temelji na robustni detekciji bazalnih dvigov kalcija ter ločeni zaznavi počasnih in hitrih oscilacij koncentracije znotrajceličnega kalcija. Predvideno je, da bo metoda omogočala neposredno primerjavo odzivov kontrolnih miši in tistih, ki nimajo izraženih receptorjev TRP3 ali TRP5, kljub slabo definiranim časovnim vrstam in prisotnosti različnih fenotipov v slednjih.

Cilj 3: Funkcija TRPM3 in TRPM5 v diabetičnih modelih.

Raziskovanje tega cilja se je v skladu s časovnico pravkar pričelo. Kljub temu smo vzpostavili model genetsko normalnih živali (kontrol) na diabetogeni dieti, opravili prvi val meritev kalcijeve aktivnosti in rezultate že v velimi meri analizirali. Torej prehitevamo realizacijo tega cilja.

Cilj 4: Razvoj matematičnega modela mrežne aktivnosti.

Raziskovanje tega cilja se v skladu s časovnico še ni pričelo. Kljub temu smo razvili večcelični model heterogenih in heterogeno sklopljenih celic, ki vključujejo aktivnost različnih ionskih kanalov, in ga uporabili za razlago časovno-prostorskega vzorca kalcijevih oscilacij v otočku, torej realnih eksperimentalnih meritev na kontrolnih živalih. V prihodnje bo potrebno prilagajanje modela meritvam na miših brez genov za TRPM3 in TRPM5, a v veliki meri prehitevamo doseganje tega cilja.

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