Raphael Rapetti-Mauss

Sig1R Protein Regulates hERG Channel Expression through a Post-translational Mechanism in Leukemic Cells

Sig1R (Sigma-1receptor) is a 25-kDa protein structurally unrelated to other mammalian proteins. Sig1R is present in brain, liver, and heart and is overexpressed in cancer cells. Studies using exogenous sigma ligands have shown that Sig1R interacts with a variety of ion channels, but its intrinsic function and mechanism of action remain unclear. The human ether-à-gogo related gene (hERG) encodes a cardiac channel that is also abnormally expressed in many primary human cancers, potentiating tumor progression through the modulation of extracellular matrix adhesive interactions. We show herein that sigma ligands inhibit hERG current density and cell adhesion to fibronectin in K562 myeloid leukemia cells. Heterologous expression in Xenopus oocytes demonstrates that Sig1R potentiates hERG current by stimulating channel subunit biosynthesis. Silencing Sig1R in leukemic K562 cells depresses hERG current density and cell adhesion to fibronectin by reducing hERG membrane expression. In K562 cells, Sig1R silencing does not modify hERG mRNA contents but reduces hERG mature form densities. In HEK cells expressing hERG and Sig1R, both proteins co-immunoprecipitate, demonstrating a physical association. Finally, Sig1R expression enhances both channel protein maturation and stability. Altogether, these results demonstrate for the first time that Sig1R controls ion channel expression through the regulation of subunit trafficking activity.

A mutation in the Gardos channel is associated with hereditary xerocytosis

The Gardos channel is a Ca(2+)-sensitive, intermediate conductance, potassium selective channel expressed in several tissues including erythrocytes and pancreas. In normal erythrocytes, it is involved in cell volume modification. Here, we report the identification of a dominantly inherited mutation in the Gardos channel in 2 unrelated families and its association with chronic hemolysis and dehydrated cells, also referred to as hereditary xerocytosis (HX). The affected individuals present chronic anemia that varies in severity. Their red cells exhibit a panel of various shape abnormalities such as elliptocytes, hemighosts, schizocytes, and very rare stomatocytic cells. The missense mutation concerns a highly conserved residue among species, located in the region interacting with Calmodulin and responsible for the channel opening and the K(+) efflux. Using 2-microelectrode experiments on Xenopus oocytes and patch-clamp electrophysiology on HEK293 cells, we demonstrated that the mutated channel exhibits a higher activity and a higher Ca(2+) sensitivity compared with the wild-type (WT) channel. The mutated channel remains sensitive to inhibition suggesting that treatment of this type of HX by a specific inhibitor of the Gardos channel could be considered. The identification of a KCNN4 mutation associated with chronic hemolysis constitutes the first report of a human disease caused by a defect of the Gardos channel.

SigmaR1 regulates membrane electrical activity in response to extracellular matrix stimulation to drive cancer cell invasiveness

The sigma 1 receptor (Sig1R) is a stress-activated chaperone that regulates ion channels and is associated with pathologic conditions, such as stroke, neurodegenerative diseases, and addiction. Aberrant expression levels of ion channels and Sig1R have been detected in tumors and cancer cells, such as myeloid leukemia and colorectal cancer, but the link between ion channel regulation and Sig1R overexpression during malignancy has not been established. In this study, we found that Sig1R dynamically controls the membrane expression of the human voltage-dependent K(+) channel human ether-à-go-go-related gene (hERG) in myeloid leukemia and colorectal cancer cell lines. Sig1R promoted the formation of hERG/β1-integrin signaling complexes upon extracellular matrix stimulation, triggering the activation of the PI3K/AKT pathway. Consequently, the presence of Sig1R in cancer cells increased motility and VEGF secretion. In vivo, Sig1R expression enhanced the aggressiveness of tumor cells by potentiating invasion and angiogenesis, leading to poor survival. Collectively, our findings highlight a novel function for Sig1R in mediating cross-talk between cancer cells and their microenvironment, thus driving oncogenesis by shaping cellular electrical activity in response to extracellular signals. Given the involvement of ion channels in promoting several hallmarks of cancer, our study also offers a potential strategy to therapeutically target ion channel function through Sig1R inhibition.

Oestrogen promotes KCNQ1 potassium channel endocytosis and postendocytic trafficking in colonic epithelium

•  Oestrogen (E2) exposure leads to a decrease in both Cl− secretion and KCNQ1 current. This inhibition is maintained by a rapid and sustained retrieval of the channel from the plasma membrane. •  The E2‐stimulated internalization of KCNQ1 occurs via a dynamin‐ and clathrin‐dependent mechanism. •  KCNQ1 is recycled back to the cell membrane via Rab4 and Rab11 rather than being degraded. •  The signalling pathway activated by E2 and leading to KCNQ1 internalization involves a signalling cascade, in which the activation of protein kinase Cδ induces the phosphorylation of AMP‐dependent kinase. Oestrogen stimulated an increase in the association of KCNQ1 with the ubiquitin ligase Nedd4.2. •  The findings provide evidence for a hormone‐stimulated regulation of KCNQ1 surface density in colonic epithelium. Moreover, this study complements the understanding of the mechanisms for E2‐induced inhibition of KCNQ1 previously described, and provides new insights on hormonal regulation of ion channel retrieval from the plasma membrane.

Sexual dimorphism and oestrogen regulation of KCNE3 expression modulates the functional properties of KCNQ1 K⁺ channels.

Non‐Technical Summary  High levels of oestrogen are known to cause fluid retention in fertile females. It is thought that the increase in body fluid volume is necessary for proper implantation of the fertilised egg in the uterus. We show that the activity of a potassium ion channel, which drives salt and water movement across the cell membranes of the intestine, is inhibited by oestrogen and this effect is only found in females and is maximal during the peak phase of oestrogen in the oestrous cycle (when fertilization and implantation occur). These findings help us to understand the molecular mechanisms underlying the fluid retention effects of oestrogen in health and the potential adverse effects this response may have in exacerbating disease where fluid secretion is compromised such as in cystic fibrosis (the so‐called CF ‘gender gap’).

Senicapoc: a potent candidate for the treatment of a subset of Hereditary Xerocytosis caused by mutations in the Gardos channel.

Hereditary xerocytosis (HX) is a form of stomatocytosis, a red blood cell disease characterized by impaired cation permeability and hemolytic anemia.[1][1] In HX, red blood cell (RBC) K+ permeability is largely increased compared to Na+ permeability thus resulting in a loss of KCl accompanied by

Bidirectional KCNQ1:β-catenin interaction drives colorectal cancer cell differentiation.

Significance The K+ channel KCNQ1 has been proposed as a tumor suppressor in colorectal cancer (CRC), but nothing is known about its regulatory role in early disease stages. KCNQ1 is a target gene of Wnt/β-catenin, which is tonically activated in CRC. We demonstrate a bidirectional interaction between KCNQ1 and β-catenin as a key regulator of CRC cell differentiation, proliferation, and invasion. KCNQ1 stabilizes β-catenin at adherent junctions to maintain an epithelial phenotype. The β-catenin:T-cell factor (TCF)-4 transcriptional pathway directly represses KCNQ1 expression, and the loss of KCNQ1 was associated with an epithelial–mesenchymal transition. The KCNQ1:KCNE3 ion channel complex expression in primary tumors was correlated with good survival outcome for patients with CRC. KCNQ1 is a potential early prognostic biomarker for CRC. The K+ channel KCNQ1 has been proposed as a tumor suppressor in colorectal cancer (CRC). We investigated the molecular mechanisms regulating KCNQ1:β-catenin bidirectional interactions and their effects on CRC differentiation, proliferation, and invasion. Molecular and pharmacologic approaches were used to determine the influence of KCNQ1 expression on the Wnt/β-catenin signaling and epithelial-to-mesenchymal transition (EMT) in human CRC cell lines of varying stages of differentiation. The expression of KCNQ1 was lost with increasing mesenchymal phenotype in poorly differentiated CRC cell lines as a consequence of repression of the KCNQ1 promoter by β-catenin:T-cell factor (TCF)-4. In well-differentiated epithelial CRC cell lines, KCNQ1 was localized to the plasma membrane in a complex with β-catenin and E-cadherin. The colocalization of KCNQ1 with adherens junction proteins was lost with increasing EMT phenotype. ShRNA knock-down of KCNQ1 caused a relocalization of β-catenin from the plasma membrane and a loss of epithelial phenotype in CRC spheroids. Overexpression of KCNQ1 trapped β-catenin at the plasma membrane, induced a patent lumen in CRC spheroids, and slowed CRC cell invasion. The KCNQ1 ion channel inhibitor chromanol 293B caused membrane depolarization, redistribution of β-catenin into the cytosol, and a reduced transepithelial electrical resistance, and stimulated CRC cell proliferation. Analysis of human primary CRC tumor patient databases showed a positive correlation between KCNQ1:KCNE3 channel complex expression and disease-free survival. We conclude that the KCNQ1 ion channel is a target gene and regulator of the Wnt/β-catenin pathway, and its repression leads to CRC cell proliferation, EMT, and tumorigenesis.

Red blood cell Gardos channel (KCNN4):the essential determinant of erythrocyte dehydration in Hereditary Xerocytosis

Recent advances have been made in the identification of molecular determinants of the rare hemolytic disease, dehydrated hereditary stomatocytosis (DHSt), also called hereditary xerocytosis (HX). This well-known red blood cell (RBC) pathology is characterized by an abnormal cation leak resulting in KCl loss and red blood cell dehydration. It leads to cell fragility and hemolytic anemia. Two different genes have been linked to this phenotype: PIEZO1 and KCNN4 coding, respectively, for Piezo1, a non-selective cation channel activated by mechanical forces, and a calcium activated K channel (KCNN4) also known the Gardos channel in RBCs. So far, three different point mutations in KCNN4 have been linked to HX. 6 The mutated KCNN4 is over-activated in patient RBCs leading to an increased K loss and, hence, water loss and cell dehydration. About a dozen mutations in Piezo1 are linked to HX, and some of them have been shown to change the kinetics of channel gating. In normal RBCs, Piezo1 appears to be a major factor in cell response to mechanical stress (by controlling calcium influx), and there is a functional connection between Piezo1 and KCNN4 through the modification of intracellular calcium concentration. Our present study was designed to evaluate in HX the functional link between mutated Piezo1 and KCNN4 and to assess the efficiency of a KCNN4 blocker, Senicapoc, to treat HX whatever the molecular cause. Our study focused on three independent index cases with a typical HX clinical and biological phenotype (Online Supplementary Table S1 and Figure 1); two were unreported cases (patients 1 and 2), and one was previously described (patient 3). Patient 1 was a 35-year-old man presenting with undiagnosed compensated hemolytic anemia and iron overload. He was investigated due to an unexplained fatal hydrops history during his wife’s second pregnancy; his first son was well and unaffected. Patient 2 was a 38-year-old women investigated for undiagnosed compensated hemolytic anemia. PIEZO1 sequencing for patient 1 and 2 revealed two new missense mutations : a c.1792G>A mutation in exon 14

Primary red cell hydration disorders: Pathogenesis and diagnosis

Hydration status is critical for erythrocyte survival and is mainly determined by intracellular cation content. Active pumps, passive transporters, and ion channels are the key components of volume homeostasis, whereas water passively fits ionic movements. Whenever cation content increases, erythrocyte swells, whereas it shrinks when cation content decreases. Thus, inappropriate cation leak causes erythrocyte hydration disorders, hemolytic anemia, and characteristic red cell shape abnormalities named stomatocytosis. All types of stomatocytosis either overhydrated or dehydrated are linked to inherited or de novo mutations in genes encoding ion transporters or channels. Although intracellular ion content can be assessed by experimental methods, laboratory diagnosis is guided by a combination of red blood cell parameters and deformability measurement when possible, and confirmed by sequencing of the putative genes. A better knowledge of the mechanisms underlying erythrocyte hydration imbalance will further lead to therapeutic improvements.

Sigma 1 Receptor and Ion Channel Dynamics in Cancer

SigmaR1 is a multitasking chaperone protein which has mainly been studied in CNS physiological and pathophysiological processes such as pain, memory, neurodegenerative diseases (amyotrophic lateral sclerosis , Parkinsons and Alzheimers diseases, retinal neurodegeneration ), stroke and addiction . Strikingly, G-protein and ion channels are the main client protein fami lies of this atypical chaperone and the recent advances that have been performed for the last 10 years demonstrate that SigmaR1 is principally activated following tissue injury and disease development to promote cell survival. In this chapter, we synthesize the data enhancing our comprehension of the interaction between SigmaR1 and ion channels and the unexpected consequences of such functional coupling in cancer development. We also describe a model in which the pro-survival functions of SigmaR1 observed in CNS pathologies are hijacked by cancer cells to shape their electrical signature and behavior in response to the tumor microenvironment .