Activation of the mechanosensitive channel PIEZO1 in the red blood cell (RBC) membrane induces a calcium influx responsible for the activation of a KCNN4 potassium channel. The functional coupling between these two ion channels leads to a leak of K, Cl and water and therefore dehydration of the RBCs. Independent mutations on either of these ion channels lead to erythrocyte dehydration responsible for the rare hemolytic anemia, hereditary dehydrated stomatocytosis.
In this project, we want to analyze the coupling between PIEZO1 and KCNN4, identify partners likely to regulate their functioning in order to propose avenues of therapeutic investigation to prevent hemolytic anemias associated with mutations in PIEZO1 or KCNN4.
This project is a multidisciplinary approach combining the genetic analysis of new mutations of KCNN4 and PIEZO1 (on patients with pathologies of the RBC membrane), with a biochemical and functional analysis of these mutations and with the bioinformatic analysis of the interactions between these channels. We want to identify:
1 / the domains of KCNN4 and PIEZO1 which interact,
2 / other proteins likely to be involved in the interaction between KCNN4 and PIEZO1,
3 / the means to act on this interaction,
4 / molecular targets for treating DHSt.
We characterized new mutations in the PIEZO1 and KCNN4 channel in patients with hemolytic anemia.
We have studied the properties of PIEZO1 carrying mutations on the N terminal part of the protein, in a structurally poorly defined area. We have shown that these mutants had different behaviors depending on whether they were in the membrane of the red blood cell or in the membrane of HEK293T cells, raising the limits of heterologous expression systems to study these ion channels and reinforcing the importance of erythrocyte context for the functioning of the couple PIEZO1 / KCNN4.
From a bioinformatics point of view, we built a complete model of the KCNN4 channel coupled to calmodulin which had never been done before.
The completion of the project will have an impact for the development of drugs for the treatment of DHSt, for the development of diagnostic tools, for the knowledge of molecular interactions between proteins and for the transmission of know-how in erythrocyte pathophysiology.
1. « Hereditary Xerocytosis : differential behavior of PIEZO1 mutations in the N-terminal extracellular domain between red blood cells and HEK cells” Yamaguchi Y. et al. Frontiers, in press
2. « Caractérisation fonctionnelle de mutations N-terminales du canal PIEZO1 dans les globules rouges et dans les cellules HEK293T » Allegrini B. et al. Communication orale au congrès de la Société Française d’Hématologie, Paris, 9-11 septembre 2021
3. KCNN4 : insights into molecular activation of a K+ channel complexed to Calmodulin, S. Jedele, C. Etchebest, SFB-GEM MEETING 2021, BIOPHYSICS OF MEMBRANES AND BEYOND, 27-30 September 2021, online, Poster
4. Potassium transport in KCNN4: investigating complete models of the membrane channel, S. Jedele, C. Etchebest, APPICOM-2021 2nd Plenary Session,Lyon, November 15-18th, 2021, Poster.
This project aims at understanding the role of the interaction between PIEZO1 a mechanically activated ion channel and KCNN4 a Ca2+ activated K+ channel in the pathophysiology of a rare red blood cell (RBC) disorder, the dehydrated Hereditary Stomatocytosis (DHSt). Patients with DHSt suffer from haemolytic anaemia and their RBCs are dehydrated due to an excess loss of KCl. The phenotype is highly heterogeneous from asymptomatic to severe form. While mutations in either PIEZO1 or KCNN4 have been associated to DHSt, the consequences of the functional interaction between both ion channels are poorly understood. We have previously shown that RBC dehydration in DHSt is due to the hyperactivity of KCNN4, whatever, the mutated channel KCNN4 or PIEZO1. Thus, there is a functional coupling between both channels and our preliminary results show also a molecular interaction between these two channels. We hypothesize that these interactions play a key role in RBC ion and water homeostasis, thus controlling RBC deformability. We propose that the different single point mutations identified in DHSt impairs interactions between the two channels, which contributes to DHSt phenotype variability.
This proposal is an interdisciplinary approach combining genetic characterization of new mutations from a collection of patient blood samples, biochemical and functional characterization of the different mutants and bioinformatics analysis of the interaction to identify:
1/the interacting parts of the two channels,
2/the other proteins likely involved in the interaction,
3/ways to modify the interaction,
4/relevant targets to treat DHSt.
Madame Hélène Guizouarn (Institut de biologie de Valrose)
The author of this summary is the project coordinator, who is responsible for the content of this summary. The ANR declines any responsibility as for its contents.
HEMATIM Hématopoïèse et immunologie
MMG Centre de Génétique Médicale de Marseille (Marseille Medical Genetics)
IBV Institut de biologie de Valrose
BIGR Biologie intégrée du globule rouge
HEMATIM LNPC / HEMATIM - UR UPJV 4666
Help of the ANR 488,093 euros
Beginning and duration of the scientific project: February 2020 - 42 Months