The Chloride Conductance Inhibitor NS3623 Enhances the Activity of a Non-selective Cation Channel in Hyperpolarizing Conditions

Abstract

Handbooks of physiology state that the strategy adopted by red blood cells (RBCs) to preserve cell volume is to maintain membrane permeability for cations at its minimum. However, enhanced cation permeability can be measured and observed in specific physiological and pathophysiological situations such as in vivo senescence, storage at low temperature, sickle cell anemia and many other genetic defects affecting transporters, membrane or cytoskeletal proteins. Among cation pathways, cation channels are able to dissipate rapidly the gradients that are built and maintained by the sodium and calcium pumps. These situations are very well-documented but a mechanistic understanding of complex electrophysiological events underlying ion transports is still lacking. In addition, non-selective cation (NSC) channels present in the RBC membrane have proven difficult to molecular identification and functional characterization. For instance, NSC channel activity can be elicited by Low Ionic Strength conditions (LIS): the associated change in membrane potential triggers its opening in a voltage dependent manner. But, whereas this depolarizing media produces a spectacular activation of NSC channel, Gárdos channel-evoked hyperpolarization's have been shown to induce sodium entry through a pathway thought to be conductive and termed Pcat. Using the CCCP method, which allows to follow fast changes in membrane potential, we show here (i) that hyperpolarization elicited by Gárdos channel activation triggers sodium entry through a conductive pathway, (ii) that chloride conductance inhibition unveils such conductive cationic conductance, (iii) that the use of the specific chloride conductance inhibitor NS3623 (a derivative of Neurosearch compound NS1652), at concentrations above what is needed for full anion channel block, potentiates the non-selective cation conductance. These results indicate that a non-selective cation channel is likely activated by the changes in the driving force for cations rather than a voltage dependence mechanism per se.

Altered Ca2+ Homeostasis in Red Blood Cells of Polycythemia Vera Patients Following Disturbed Organelle Sorting during Terminal Erythropoiesis

“…Automated patch-clamp on the SyncroPatch 384PE (Nanion Technologies, Munich, Germany) was used to measure Gárdos activity, with a modified protocol previously described [ 14 ]. Briefly, after three washes with PBS and upon cell catch, external recording solution was added to the wells followed by 10 µM NS3623 (a powerful chloride channel inhibitor [ 15 ]), 10 µM NS309 (Alomone labs, Jerusalem, Israel), 5 µM TRAM-34 (a specific Gárdos channel inhibitor, Tocris, Bristol, UK), and 30 µM GdCl 3 (a generic non selective cation channel inhibitor, Sigma-Aldrich, St. Louis, MO, USA). Internal solution (in mM): 10 KCl, 110 KF, 10 NaCl, 10 EGTA, and 10 HEPES/KOH (pH 7.2).…”

Dual action of Dooku1 on PIEZO1 channel in human red blood cells

“…It has been widely reported that the activation of PIEZO1 by Yoda1 induces a cell dehydration accompanied by a reversal of sodium and potassium gradients due to its nature of being a non-selective cation channel ( Monedero Alonso et al, 2021 ; Pérès et al, 2021 ).…”

On the involvement of cell volume regulation mechanisms in post-hypertonic lysis and slow-freezing injury of human erythrocytes and its broader cryobiological significance

“…27,44,45 Specifically to human RBCs, experimental studies also support the involvement of ion transport pathways in the osmotic response of RBCs as the ion flux rates may be high enough to result in considerable KCl loss within ~15 minutes 67 (action equivalent to RVD response) or increase of intracellular Na in 30 minutes (RVI). 50 Furthermore, wholecell recordings of 68 to 500 pico-siemens cation conductances were reported, 51,68,69 corresponding to about a 3 to 4 order increase compared to the normal isotonic value. 70 This experimental observation supports the relatively high rate of predicted NaCl loading occurring only via physiological ion transport pathways (consult Figure 6).…”

“…48 The presence of shrinkage-activated cation channels (classified as HICCs) in the RBC plasma membrane has been confirmed. 49 The capacity of human RBCs to substantially increase the cation permeability was reported several times [49][50][51][52] with even several-order increases being observed. 53 It is suggested here that HICCs might be a major contributor to the RVI response, which is hypothesized to cause sodium influx (coupled with an influx of chlorine ions) under hypertonic conditions -emphasizing the physiological route for the salt loading.…”

“…61 All parameters used in this study (Table 1 and Table 2) correspond to the human RBC state parameters at normal isotonic conditions, consistent with previous studies. 36,50,62,63 The intracellular concentration of non-permeants is assumed to be 50 mmol/L with average charge valency of -1 (indicating negative charge). 61, 64 The only parameter that is varied in this study is the cation permeability -the quantitative consequence of the hypothesized increased permeability due to the activation of ion transport pathways facilitating acute cell volume regulation.…”