Összes szerző

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Csanády László

az alábbi absztraktok szerzői között szerepel:

Bartók Ádám
Molecular strategies for heat detection by TRPM2 channels

Aug 30 - szerda

09:20 – 09:40

Membránok és membránfehérjék biofizikája

E22

Molecular strategies for heat detection by TRPM2 channels

Ádám Bartók¹, László Csanády¹

¹ Semmelweis University, Department of Biochemistry

TRPM2, a Ca²⁺ permeable non-selective cation channel co-activated by cytosolic Ca²⁺ and ADP ribose (ADPR), plays key role in the central regulation of body temperature. The central thermostat must contain a temperature sensor, which is able to differentiate temperature fluctuations as small as ±1°C around 37°C. To address whether heat-activation of TRPM2 gating in intact cells is an intrinsic property of the TRPM2 protein, temperature dependence of TRPM2 currents was studied in inside-out patches between 15°C and 40°C, across broad ranges of concentrations of both agonists. For fully liganded TRPM2 pore opening is intrinsically endothermic, the enthalpy of opening is ~180 kJ/mol. However, the TRPM2 temperature threshold is too high (>40°C) for unliganded, but too low (<15°C) for fully liganded channels. Calculations based on a mechanistic gating model indicate that TRPM2 warmth sensitivity around 37°C is restricted to narrow ranges of agonist concentrations. For ADPR that range (submicromolar-to-micromolar) matches, but for Ca²⁺ (>1 µM) it exceeds bulk cytosolic values, suggesting that a Ca²⁺nanodomain drives TRPM2 activation in vivo. TRPM2 is Ca²⁺ permeable, and the binding sites for activating Ca²⁺ are near the cytosolic pore entrance. We therefore investigated how the presence of a physiological extracellular [Ca²⁺] affects temperature dependence of TRPM2 gating between 37°C and 40°C, while bulk cytosolic [Ca²⁺] was buffered to 100 nM and [ADPR] was set to 2 µM. Under such quasi-physiological conditions Po was ~0.04 and ~0.34, respectively, at 37°C and 40°C. These findings demonstrate and quantitate the positive feedback provided by Ca²⁺ influx. The larger Po at 40°C elevates local [Ca²⁺] around the activating sites, which in turn further enhances Po. That positive feedback provides strong amplification to the TRPM2 temperature response (Q10~1000), enabling the TRPM2 protein to autonomously respond to tiny temperature fluctuations around 37°C. [1].

Acknowledgment

Project was supported by MTA Lendület grant LP2017-14/2017 to L.C. Á.B. was supported by the János Bolyai Research Scholarship of the Hungarian Academy of Sciences (BO/00103/20)

References

[1] Bartok A, Csanady L (2022) PNAS

Tóth Ádám Viktor
Molecular characterization of pathological and tissue-specific TRPM2 cation channel variants

Aug 30 - szerda

15:30 – 17:00

II. Poszterszekció

P57

Molecular characterization of pathological and tissue-specific TRPM2 cation channel variants

Ádám V. Tóth¹, Ádám Bartók¹ and László Csanády¹

¹ Department of Biochemistry, Semmelweis University, Budapest, Hungary

TRPM2 is a temperature-sensitive, Ca2+-permeable, non-selective cation channel, showing high level of expression in cells of the central nervous system, bone marrow, granulocytes and pancreatic β-cells. Activation of the channel requires the simultaneous intracellular presence of adenosine diphosphate ribose (ADPR), Ca2+ ions and phosphatidylinositol 4,5-bisphosphate (PIP2). In these cells, TRPM2 contributes to Ca2+ influx resulting in important physiological and pathological functions, such as body temperature regulation, cytokine production, oxidative stress response, inflammation or controlled cell death. Moreover, certain TRPM2 point mutations show close genetic connection with bipolar disorder (D543E, R755C) [1] or amyotrophic lateral sclerosis and Parkinson's dementia (P1018L) [2]. Interestingly, alternative splice products were isolated from healthy neutrophil granulocytes (ΔC-TRPM2) [3] and from striatum (SSF-TRPM2) [4], which presumably modify the ligand specificity and function of the channel in a cell-specific manner.

Until now, the mentioned variants have only been investigated using fluorescent imaging techniques or whole-cell electrophysiological methods providing limited opportunities to study the ligands acting intracellularly. Our aim is to examine the listed ion channel variants in molecular details. To this end, expression vector encoding TRPM2 variants have been produced and expressed transiently in HEK cells. Functional measurements are performed by inside-out patch clamp configuration enabling reliable recordings of micro- and macroscopic currents and fast exchange of intracellular ligands. With our method, it is possible to map crucial biological and biophysical parameters of the channel variants: ADPR and Ca2+ sensitivity, gating parameters, inactivation kinetics, temperature dependence. This detailed knowledge is essential for a comprehensive understanding of the role of these mutants in pathomechanisms and tissue-specific variant functions.

References

[1] A. McQuillin et al. Mol Psychiatry 11, 134-142 (2006)

[2] M. C. Hermosura et al. Proc Natl Acad Sci USA 105, 18029-34 (2008)

[3] E. Wehage et al. J Biol Chem 277, 23150-6 (2002)

[4] T. Uemura et al. Biochem Biophys Res Commun 328, 1232-43 (2005)