ORIGINAL RESEARCH published: 16 April 2019 doi: 10.3389/fphys.2019.00386 Red Blood Cell Membrane Conductance in Hereditary Haemolytic Anaemias Polina Petkova-Kirova 1 , Laura Hertz 2,3 , Jens Danielczok 2 , Rick Huisjes 4 , Asya Makhro 5 , Anna Bogdanova 5 , Maria del Mar Mañú-Pereira 6 , Joan-Lluis Vives Corrons 7 , Richard van Wijk 4 and Lars Kaestner 2,3* 1 Institute of Molecular Cell Biology, Saarland University, Homburg, Germany, 2 Theoretical Medicine and Biosciences, Saarland University, Homburg, Germany, 3 Experimental Physics, Saarland University, Saarbrücken, Germany, 4 Department of Clinical Chemistry & Haematology, University Medical Center Utrecht, Utrecht, Netherlands, 5 Red Blood Cell Research Edited by: Group, Institute of Veterinary Physiology, Vetsuisse Faculty, Zurich Center for Integrative Human Physiology (ZIHP), University Philippe Connes, of Zürich, Zurich, Switzerland, 6 Vall d’Hebron Research Institute, Vall d’Hebron University Hospital, Barcelona, Spain, 7 Red Université Claude Bernard Lyon 1, Blood Cell Defects and Hematopoietic Disorders Unit, Josep Carreras Leukaemia Research Institute, Barcelona, Spain France Reviewed by: Congenital haemolytic anaemias are inherited disorders caused by red blood cell Stéphane Egee, UMR8227 Laboratoire de Biologie membrane and cytoskeletal protein defects, deviant hemoglobin synthesis and Intégrative des Modèles Marins, metabolic enzyme deficiencies. In many cases, although the causing mutation France John Stanley Gibson, might be known, the pathophysiology and the connection between the particular University of Cambridge, mutation and the symptoms of the disease are not completely understood. United Kingdom Thus effective treatment is lagging behind. As in many cases abnormal red *Correspondence: blood cell cation content and cation leaks go along with the disease, by direct Lars Kaestner lars_kaestner@me.com electrophysiological measurements of the general conductance of red blood cells, we aimed to assess if changes in the membrane conductance could be a Specialty section: This article was submitted to possible cause. We recorded whole-cell currents from 29 patients with different Red Blood Cell Physiology, types of congenital haemolytic anaemias: 14 with hereditary spherocytosis due a section of the journal to mutations in α-spectrin, β-spectrin, ankyrin and band 3 protein; 6 patients Frontiers in Physiology with hereditary xerocytosis due to mutations in Piezo1; 6 patients with enzymatic Received: 21 January 2019 Accepted: 21 March 2019 disorders (3 patients with glucose-6-phosphate dehydrogenase deficiency, 1 patient Published: 16 April 2019 with pyruvate kinase deficiency, 1 patient with glutamate-cysteine ligase deficiency Citation: and 1 patient with glutathione reductase deficiency), 1 patient with β-thalassemia Petkova-Kirova P, Hertz L, Danielczok J, Huisjes R, Makhro A, and 2 patients, carriers of several mutations and a complex genotype. While Bogdanova A, Mañú-Pereira MdM, the patients with β-thalassemia and metabolic enzyme deficiencies showed no Vives Corrons J-L, van Wijk R and changes in their membrane conductance, the patients with hereditary spherocytosis Kaestner L (2019) Red Blood Cell Membrane Conductance and hereditary xerocytosis showed largely variable results depending on the in Hereditary Haemolytic Anaemias. underlying mutation. Front. Physiol. 10:386. doi: 10.3389/fphys.2019.00386 Keywords: haemolytic anemia, patch-clamp, electrophysiology, hereditary spherocytosis, hereditary xerocytosis Frontiers in Physiology | www.frontiersin.org 1 April 2019 | Volume 10 | Article 386 Petkova-Kirova et al. Red Cell Electrophysiology in Anaemias INTRODUCTION deficiency (G6PD), pyruvate kinase deficiency (PKD), glutamate- cysteine ligase (γ-glutamylcysteine synthetase) (GCL) deficiency, Haemolytic anaemias, characterized by the abnormal breakdown glutathione reductase deficiency and β-thalassemia. of red blood cells (RBCs), could be either acquired or inherited. The latter are a diverse group of diseases that could be classified based on the affected RBC component into membranopathies, MATERIALS AND METHODS haemoglobinopathies, and enzymopathies (Dhaliwal et al., 2004). Membranopathies are presented by hereditary spherocytosis Patients (HS), hereditary elliptocytosis (HE) and its aggravated form Patients diagnosed with different types of haemolytic anemia pyropoikilocytosis (HPP) with defective structural membrane were enrolled in the study after signing an informed consent. and cytoskeletal proteins (Iolascon et al., 2003) and by the Patients’ data were handled anonymously as outlined in the ethics largely heterogeneous group of stomatocytosis divided in a agreements. These agreements were approved by the Medical most general way, but not exhaustively, into overhydrated Ethical Research Board (MERB) of the University Medical stomatocytosis (OHSt), cryohydrocytosis (CHC) and some Center Utrecht, the Netherlands (UMCU) under reference code types of familial pseudohyperkalaemia (FP) (overhydrated 15/426M “Disturbed ion homeostasis in hereditary hemolytic RBCs) and dehydrated stomatocytosis (DHSt) (hereditary anemia” and by the Ethical Committee of Clinical Investigations xerocytosis (HX)) (dehydrated RBCs) with defective ion channels of Hospital Clinic, Spain (IDIBAPS) under reference code or transporters (Iolascon et al., 2003; Bruce et al., 2009). 2013/8436. Exclusion criteria were erythrocyte transfusion in the Haemoglobinopathies are presented by β-thalassemia (Cao and past 90 days, age below 3 years and/or bodyweight lower than Galanello, 2010) and sickle cell disease (Ware et al., 2017) with 18 kg. Blood from healthy control donors was anonymously defective hemoglobin and enzymopathies are presented most obtained using the approved medical ethical protocol of commonly by glucose-6-phosphate dehydrogenase deficiency 07/125 Mini Donor Dienst, also approved by the MERB of (G6PD) (Luzzatto et al., 2016) and pyruvate kinase deficiency UMCU. The blood of the patient/patients and the healthy (PKD) (Zanella et al., 2005) but also by glutamate-cysteine donor anti-coagulated in heparin was shipped overnight from ligase (γ-glutamylcysteine synthetase) (GCL) deficiency (Ristoff the University Medical Center Utrecht (Utrecht, Netherlands) and Larsson, 1998) and glutathione reductase deficiency (van or from Institut d’Investigacions Biomèdiques August Pi i Zwieten et al., 2014). Although much is known so far, Sunyer/Hospital Clínic de Barcelona (Barcelona, Spain) to especially regarding the defective genes related to hereditary Saarland University (Homburg, Germany) without additional haemolytic anaemias, there are still questions, whose answers cooling as previously tested/simulated (Makhro et al., 2016). would lead to a much better understanding of the disease and All patients included in the study were genetically screened possibly to a more effective treatment. A recurrent issue is for mutations by next-generation sequencing and diagnosed if a changed membrane conductance, resulting from primary with the following types of anemia: 14 patients were diagnosed mutated channels or secondary adapted ones, does contribute with HS (due to mutations in α-spectrin, β-spectrin, ankyrin to the various phenotypes. Most of the research has been done and band 3 protein), using golden standard techniques (EMA- on membranopathies, understandable, primarily on the ones binding, osmotic gradient ektacytometry and osmotic fragility linked to mutations in ion channels or transporters such as test), 6 patients were diagnosed with hereditary xerocytosis (due band 3 protein, Rh-associated glycoprotein (RhAG), the glucose to mutations in Piezo 1), 6 patients had enzymatic disorders (3 transporter GLUT1, Piezo1 and the Gardos channel (KCNN4) patients with glucose-6-phosphate dehydrogenase deficiency, 1 and accompanied by abnormal RBC cation content and disrupted patient with pyruvate kinase deficiency, 1 patient with glutamate- volume homeostasis (Badens and Guizouarn, 2016). However, cysteine ligase deficiency and 1 patient with glutathione reductase although on most occasions, the RBC cation content linked to the deficiency), 1 patient had β-thalassemia and 2 were carriers particular mutation has been extensively described (e.g., Stewart of several mutations and a complex genotype. The genotype et al., 2011 for R730C in band 3 protein or Fermo et al., 2017 of the patients with HS, HX and of the two patients with for R352H in the Gardos channel) and the defective channels, several mutations is given in Table 1. The numbering of when known, expressed and studied in heterologous systems the patients and the corresponding healthy controls is kept (e.g., Glogowska et al., 2017 for a number of Piezo1 mutations) consistent with previous research (Hertz et al., 2017) studying the with a few exceptions (Stewart et al., 2011; Andolfo et al., 2013; same patient group. Shmukler et al., 2014; Fermo et al., 2017; Rotordam et al., 2018), direct electrophysiological measurements of membrane Patch Clamp Analysis conductance in mutated RBCs have been scarce. Patch-clamp whole-cell measurements were performed with a Thus, within the CoMMiTMenT project, by direct RBCs NPC-16 Patchliner (Nanion Technologies, Munich, Germany) as electrophysiological measurements in physiological solutions, previously described (Petkova-Kirova et al., 2018). Briefly, the we aimed to investigate whether RBC membrane conductance resistance of the chips was between 5 and 8 M with internal and changes are accompanying HS due to mutations in the SPTA1 external solutions as follows (in mM): KCl 70, KF 70, NaCl 10, gene (coding for α-spectrin), SPTB (β-spectrin), ANK1 (ankyrin), HEPES 10, EGTA 3, CaCl2 1.2 to give free [Ca2+ ]i = 120 nM, and SLC4A1 (band 3 protein); HX due to mutations in PIEZO1; pH = 7.2 adjusted with KOH (internal) and NaCl 140, KCl 4, enzymatic disorders due to glucose-6-phosphate dehydrogenase MgCl2 5, CaCl2 2, D-glucose 5, HEPES 10, pH = 7.3 adjusted Frontiers in Physiology | www.frontiersin.org 2 April 2019 | Volume 10 | Article 386 Petkova-Kirova et al. Red Cell Electrophysiology in Anaemias TABLE 1 | Patients overview. Patient Clinical presentation Genotype Current compared to Current compared to transportation control general control P18.1 HS (α-spectrin) c.2755G > T (p.Glu919); αLELY Current ↑ Current ↑ P19.1 HS (α-spectrin) c.678G > A (p.Glu227fs); αLELY Current ↑ (not Current ↑ statistically significant) P15.1 HS (α-spectrin) c.4339-99C > T p.(?) No change No change P20.1 HS (α-spectrin) c.[4339-99C > T; c.4347G > T] p.[(?; No change No change Lys1449Asn)] P17.1 (splenectomized) HS (ankyrin) c.341C > T (p.Pro114Leu) Current ↓ ∗ No change P17.2 HS (ankyrin) c.341C > T (p.Pro114Leu) Current ↓ ∗ No change P11.1 HS (ankyrin) c.1943delC;c.2042delC No change No change (p.Ala648fs;p.Ala681fs) P13.1 HS (ankyrin) c.344T > C (p.Leu115Pro) No change No change P13.2 HS (ankyrin) c.344T > C (p.Leu115Pro) No change No change P14.1 HS (ankyrin) c.2559-2A > G (splicing) No change No change P10.1 HS (β-spectrin) c.2470C > T (p.Gln824) Outward current ↓↓ ∗ No change P21.1 HS (β-spectrin) c.3449G > A (p.Trp1150) No change No change P12.1 HS (band 3 protein) c.2348T > A (p.Ile783Asn) Current ↓ Inward current ↓ P16.1 HS (band 3 protein) c.2057 + 1G > A (splicing) No change No change P11.1 SPTB c.154delC; p.Arg52fs + RHAG Current ↑ Current ↑ c.808G > A; p.Val270Ile P22.1 SPTA1 c.460_462dupTTG; Current ↑ Current ↑ p.Leu154dup + PKLR c.1687G > A; p.Gly563Ser + del3.7Kb HBA Family 1 P51.3 HX c. 7367G > A p.Arg2456His No change No change P51.4 HX c. 7367G > A p.Arg2456His No change No change Family 2 P53.3 HX c. 6262C > G, p. Arg2088Gly No change No change P53.2 HX c. 6262C > G, p. Arg2088Gly No change No change Family 3 P50.2 HX c.1276T > C p. Cys426Arg No change Outward current ↑ ∗ Family 4 P52.1 HX c.7483_7488dupCTGGAG Current ↓ Current ↓ p.2495_2496dupLeuGlu P40.1 G6PD No change No change P41.1 G6PD No change No change P42.1 G6PD No change No change P43.1 GCLD No change No change P44.1 GRD No change No change P45.1 PKD No change No change P60.1 β-thalassemia No change No change An upward arrow indicates an increase in current and a downward arrow indicates a decrease in current compared to the respective control. Two arrows label an extreme change in the designated direction. Patients that show a change in current compared to both the transportation and a general control are given in bold. Asterisks mark a deviation commented on in the main text. HS, hereditary spherocytosis; SPTB, β-spectrin; SPTA1, α-spectrin; HX, hereditary xerocytosis; GCLD, glutamate-cysteine ligase deficiency; GRD, glutathione reductase deficiency; PKD, pyruvate kinase deficiency. with NaOH (external). Gigaseals were considered successful if voltage steps from −100 to 100 mV for 500 ms in 20 mV exceeding 5 G. Gigaseal formation was facilitated by the use increments at 5 s intervals, the holding potential being set at of a seal enhancing solution as recommended by the Patchliner −30 mV. Data are presented as current density (current divided manufacturer and containing (in mM): NaCl 80, KCl 3, MgCl2 10, by capacitance, the latter estimated at the time of attaining the CaCl2 35, HEPES 10, pH = 7.3 adjusted with NaOH. Whole-cell whole-cell configuration and by using a short test pulse of 10 mV, configuration was achieved by negative pressure suction pulses 5 ms) and given as means ± SEMs (n denotes number of cells and between −45 mbar and −150 mbar and its formation judged by N – number of patients). Significant differences are determined the appearance of sharp capacitive transients. Whole-cell patch- based on an unpaired t-test and Welch’s correction for unequal clamp recordings were conducted at room temperature using variances, when needed, with ∗ denoting p < 0.05. Frontiers in Physiology | www.frontiersin.org 3 April 2019 | Volume 10 | Article 386 Petkova-Kirova et al. Red Cell Electrophysiology in Anaemias RESULTS vs. 0.69 pA/pF general control; p > 0.05). However, the two patients, heterozygous for the SPTA1 mutation and carrying at Whole-cell patch clamp measurements were performed to assess the same time an αLELY allele showed an increase in their inward possible differences in the membrane conductance of hereditary current (Figure 3). Figures 3Aa,Ba consider the particular SPTA1 anemia patients compared to healthy controls. Regarding αLELY patients [patient P18.1 (10 cells) and patient P19.1 (6 controls, we have compared the currents measured from the cells), respectively] vs. their transportation controls [C18 (6 cells) RBCs of our patients once with their transportation control, i.e., and C19 (7 cells), respectively]. Figures 3Ab,Bb compare the currents measured from the RBCs of a healthy subject, whose particular SPTA1 αLELY patients [P18.1 (10 cells) and P19.1 (6 blood was delivered together with the blood of the patient, and cells) vs. a control pooled over all the cells of all healthy subjects once with a general, pooled, control, i.e., currents measured from included in the study (175 cells)]. Figures 3Ac,Ad,Bc,Bd present the cells of all healthy subjects delivered throughout the study raw current traces recorded from the RBCs of a healthy subject (Table 1). The rational for this ‘double comparison’ is provided (Figures Ac,Bc), P18.1 (Figure 3Ad), and P19.1 (Figure 3Bd). in the discussion. Throughout the whole study, accordingly in None of the two patients showed any difference in capacitance the manuscript, the abbreviation “P” stands for patient and “C” compared with the general or with its transportation control stands for a control, healthy subject. (0.59 pA/pF P18.1 vs. 0.74 pA/pF C18, p > 0.05; 0.59 pA/pF P18.1 vs. 0.69 pA/pF general control, p > 0.05; 0.66 pA/pF P19.1 Hereditary Spherocytosis vs. 0.58 pA/pF C19, p > 0.05; 0.66 pA/pF P19.1 vs. 0.69 pA/pF Studied were patients with pathogenic mutations in SPTA1 general control, p > 0.05). Furthermore, while HS patients with (α-spectrin) (4 patients: P15.1, P18.1, P19.1, P20.1), SPTB underlying defects in ANK1 (6 patients) showed no significant (β-spectrin) (2 patients: P10.1 and P21.1), ANK1 (ankyrin) differences neither in their currents, nor in their capacitances (6 patients: P11.1, P13.1, P13.2, P14.1, P17.1, and P17.2), (0.67 pA/pF patients vs. 0.63 pA/pF transportation control, and SLC4A1 (band 3 protein) (2 patients: P12.1 and P16.1), p > 0.05; 0.67 pA/pF patients vs. 0.69 pA/pF general control; whose blood was delivered and, respectively, recorded from p > 0.05) compared to the control group [Figure 4Aa considered together with the blood of a healthy subject (transportation are the control healthy subjects delivered together with the control). While no changes in the membrane conductance, patients (4 healthy subjects) and Figure 4Ab considered are all nor in membrane capacitance (0.69 pA/pF general control healthy subjects included in the study (27 healthy subjects)], there vs. 0.63 pA/pF patients, p > 0.05; 0.65 pA/pF transportation was a family of patients P17.1 (splenectomized) and P17.2 (with control vs. 0.63 pA/pF patients; p > 0.05) were revealed with spleen) in whom an ANK1 mutation [c.341C > T (p.Pro114Leu)] patients taken altogether (Figure 1), differences were observed was associated with a decreased membrane conductance when in certain patients’ groups as well as linked to particular compared to their own controls (Figures 4Ba,Ca, respectively) mutations Figures 3–5). Thus patients with mutations in SPTA1 but not when compared to the pooled control of all healthy cells (4 patients), showed no significant differences compared to (Figures 4Bb,Cb, respectively). Comparison of the capacitances healthy controls delivered at the same days (4 healthy subjects) of P17.1 and P17.2 with their controls as well as the capacitance (Figure 2A) or compared to a control pooled over all healthy of C17 with the general control is as follows: 0.67 pA/pF P17.1 subjects included in the study (27 healthy subjects) (Figure 2B). vs. 0.525 pA/pF C17, p > 0.05; 0.67 pA/pF P17.1 vs. 0.69 pA/pF Capacitances were not different either (0.67 pA/pF patients vs. general control, p > 0.05; 0.62 pA/pF P17.2 vs. 0.525 pA/pF 0.68 pA/pF transportation control, p > 0.05; 0.67 pA/pF patients C17, p > 0.05; 0.62 pA/pF P17.2 vs. 0.69 pA/pF general control, FIGURE 1 | Whole-cell recordings of ion currents from RBCs of healthy donors and HS patients. Compared are the I/V-curves of all HS patients (N = 14) with the I/V curves of their transportation controls (N = 12) (A) and with the I/V curves of all healthy subjects delivered throughout the study (N = 27) (B), where N denotes number of healthy subjects or HS patients. No changes were observed in capacitance either with the transportation (0.63 pA/pF patients vs. 0.65 pA/pF transportation control; p > 0.05) or with the general control (0.63 pA/pF patients vs. 0.69 pA/pF general control; p > 0.05). Currents were elicited by voltage steps from –100 to 100 mV for 500 ms in 20 mV increments at V h = –30 mV. Data are expressed as mean current density ± SEMs. Frontiers in Physiology | www.frontiersin.org 4 April 2019 | Volume 10 | Article 386 Petkova-Kirova et al. Red Cell Electrophysiology in Anaemias FIGURE 2 | Whole-cell recordings of ion currents from RBCs of healthy donors and HS patients with α-spectrin mutations. Compared are the I/V-curves of all HS patients with α-spectrin mutations (N = 4) with the I/V curves of their own transportation controls (N = 4) (A) and with the I/V curves of all healthy subjects delivered throughout the study (N = 27) (B), where N denotes the number of healthy subjects or HS patients. No changes were observed in capacitance either with the transportation (0.67 pA/pF patients vs. 0.68 pA/pF control; p > 0.05) or with the general control (0.67 pA/pF patients vs. 0.69 pA/pF control; p > 0.05). Currents were elicited by voltage steps from –100 to 100 mV for 500 ms in 20 mV increments at V h = –30 mV. Data are expressed as mean current density ± SEMs. FIGURE 3 | Whole-cell recordings of ion currents from RBCs of healthy donors and HS patients with α-spectrin mutations and carrying at the same time an αLELY allele. Compared are the I/V curves of P18.1 (n = 10) with its own transportation control C18 (n = 6) (Aa) as well as with a general control (n = 175) (Ab), where n denotes the number of cells from the patient or the controls. As examples raw current traces recorded from the RBCs of a healthy donor (C18), whose blood was delivered together with the blood of P18.1 (Ac) and of patient P18.1 (Ad) are presented. Capacitances were not any different either with the transportation control (0.59 pA/pF P18.1 vs. 0.74 pA/pF C18; p > 0.05) or with the general control (0.59 pA/pF patient vs. 0.69 pA/pF control; p > 0.05). Compared are the I/V curves of P19.1 (n = 6) with its own transportation control C19 (n = 7) (Ba) as well as with a general control (n = 175) (Bb), where n denotes the number of cells from the patient or the controls. As examples raw current traces recorded from the RBCs of a healthy donor (C19), whose blood was delivered together with the blood of P19.1 (Bc) and of patient P19.1 (Bd) are presented. Capacitances were not any different either with the transportation control (0.66 pA/pF P19.1 vs. 0.58 pA/pF C19; p > 0.05) or with the general control (0.66 pA/pF patient vs. 0.69 pA/pF general control; p > 0.05). Significant differences are determined based on an unpaired t-test with ∗ representing p < 0.05. Mutations below patients numbers are designated as amino acid substitutions in the respective protein. The label αLELY next to the mutation stands for the presence of an αLELY allele in the corresponding patient. Frontiers in Physiology | www.frontiersin.org 5 April 2019 | Volume 10 | Article 386 Petkova-Kirova et al. Red Cell Electrophysiology in Anaemias FIGURE 4 | Whole-cell recordings of ion currents from RBCs of healthy donors and HS patients. Whole-cell recordings of ion currents from RBCs of healthy donors and HS patients with ankyrin mutations (A–C). Compared are the I/V-curves of all HS patients with ankyrin mutations (N = 6) with the I/V curves of their own transportation controls (N = 4) (Aa) and with the I/V curves of all healthy subjects delivered throughout the study (N = 27) (Ab), where N denotes the number of healthy subjects or HS patients. No changes were observed in capacitance either with the transportation (0.67 pA/pF patients vs. 0.63 pA/pF transportation control; p > 0.05) or with the general control (0.67 pA/pF patients vs. 0.69 pA/pF general control; p > 0.05). Compared are the I/V curves of P17.1 (n = 13) and its own transportation control C17 (n = 6) (Ba) and the I/V curves of P17.2 (n = 11) with its own transportation control C17 (n = 6) (Ca), where n denotes the number of cells from the patient or the control. (Bb,Cb) Compare the I/V curve of a general control based on currents recorded from all cells of all healthy subjects delivered throughout the study (n = 175) with the I/V curve of P17.1 (n = 13) (Bb) and P17.2 (n = 11) (Cb) (n denotes number of cells). (Continued) Frontiers in Physiology | www.frontiersin.org 6 April 2019 | Volume 10 | Article 386 Petkova-Kirova et al. Red Cell Electrophysiology in Anaemias FIGURE 4 | Continued Both patients 17.1 and 17.2 showed no differences in capacitance either with their transportation or with the general control (0.67 pA/pF P17.1 vs. 0.525 pA/pF C17, p > 0.05; 0.67 pA/pF P17.1 vs. 0.69 pA/pF general control, p > 0.05; 0.62 pA/pF P17.2 vs. 0.525 pA/pF C17, p > 0.05; 0.62 pA/pF P17.2 vs. 0.69 pA/pF general control, p > 0.05). However, a difference was uncovered between C17 and the general control (0.525 pA/pF C17 vs. 0.69 pA/pF general control; p < 0.05). Whole-cell recordings of ion currents from RBCs of a healthy donor and a HS patient with band 3 protein mutation (D). Compared are the I/V curves of P12.1 (n = 17) with its own transportation control C12 (n = 7) (Da) as well as with a general control (n = 175) (Db), where n is the number of cells of the patient or the controls. No changes were observed in capacitance either with the transportation (0.65 pA/pF P12.1 vs. 0.59 pA/pF C12; p > 0.05) or with the general control (0.65 pA/pF P12.1 vs. 0.69 pA/pF general control; p > 0.05). Currents were elicited by voltage steps from –100 to 100 mV for 500 ms in 20 mV increments at V h = –30 mV. Data are expressed as mean current density ± SEMs. Significant differences are determined based on an unpaired t-test with ∗ representing p < 0.05. Mutations below patients numbers are designated as amino acid substitutions in the respective protein. p > 0.05; 0.525 pA/pF C17 vs. 0.69 pA/pF general control; C53, C50, and C52, respectively). No changes in the membrane p < 0.05. Moreover a patient with a band 3 protein mutation conductance or in the membrane capacitance were revealed [SLC4A1 (2348T > A, Ile783Asn), P12.1 showed a decreased with patients taken altogether (Figure 7A) as well as in two current compared to its own, transportation, control (C12) families (Family1 (P51.3 and P51.4) and Family 2 (P53.2 and to a pooled general control (Figures 4Da,b, respectively)]. and P53.3) compared both to their transportation controls No difference was found when the capacitance of the patient and to a general control (Figures 7Ba,b,Ca,b, respectively). was compared with that of the general or the transportation However, P50.2 (Family 3), although showing no difference control (0.65 pA/pF P12.1 vs. 0.59 pA/pF C12, p > 0.05; with its transportation control (Figure 8Aa), demonstrated 0.65 pA/pF P12.1 vs. 0.69 pA/pF general control, p > 0.05). increased conductance compared to the general, pooled, control Out of the two patients with SPTB mutations (P10.1 and P21.1) (Figure 8Ab). There was also a family (Family 4 with P52.1) (Figures 5A,Ba,b) one patient, P10.1, showed a significantly in which the Piezo1 mutation (c.7483_7488dupCTGGAG different conductance compared to its transportation control C10 p.2495_2496dupLeuGlu) was associated with a decreased (Figure 5Ba). However, based on the fact that P10.1 showed conductance compared both to the transportation and to no difference with the general control (Figure 5Bb) and that the general, pooled, control (Figures 8Ba,b, respectively). P21.1 showed no difference either with its transportation control Both Family 3 and Family 4 did not show a change in (Figure 5Aa) or with the general control (Figure 5Ab) as well their capacitance compared to their transportation or to as on the fact that C10 is very different from the general, pooled the general control. control (an outlier according to the Grubbs’ test) (Figure 5Bc), we conclude that patients with β-spectrin mutations show no Enzymopathies changes in their current. Comparison of capacitances of P21.1 Considered were 6 patients with enzymatic disorders as and P10.1 with their controls as well as the capacitance of C10 follows: 3 patients with glucose-6-phosphate dehydrogenase with the general control is as follows: 0.63 pA/pF P21.1 vs. deficiency (P40.1, P41.1, and P42.1), 1 patient with pyruvate 0.69 pA/pF C21, p > 0.05; 0.63 pA/pF P21.1 vs. 0.69 pA/pF kinase deficiency (P45.1), 1 patient with glutamate-cysteine general control, p > 0.05; 0.68 pA/pF P10.1 vs. 0.82 pA/pF ligase deficiency (P43.1) and 1 patient with glutathione C10, p < 0.05; 0.68 pA/pF P10.1 vs. 0.69 pA/pF general control, reductase deficiency (P44.1). None of the patients showed p > 0.05; 0.82 pA/pF C10 vs. 0.69 pA/pF general control; p < 0.05. a difference in their membrane conductance or capacitance C10 shows a significantly increased capacitance compared to compared to a general or their own transportation P10.1 as well as to the general control. control (Figures 9, 10). Two additional patients P11.1 and P22.1, carriers of several mutations and a complex genotype (SPTB c.154delC; Beta-Thalassemia p.Arg52fs + RHAG c.808G > A; p.Val270Ile and SPTA1 The patient (P60.1) with β-thalassemia showed no difference c.460_462dupTTG; p.Leu154dup + PKLR c.1687G > A; in its membrane conductance or membrane capacitance p.Gly563Ser + del3.7Kb HBA, respectively) show an increase either compared to a general or to its own, transportation, in their currents (Figures 6A,B, respectively). The capacitances control (Figure 11). of none of the patients show any difference with their controls (0.59 pA/pF P11.1 vs. 0.66 pA/pF C11, p > 0.05; 0.59 pA/pF P11.1 vs. 0.69 pA/pF general control, p > 0.05; 0.61 pA/pF P22.1 DISCUSSION vs. 0.64 pA/pF C22, p > 0.05; 0.61 pA/pF P22.1 vs. 0.69 pA/pF general control, p > 0.05). Hereditary Spherocytosis Electrophysiological measurements revealed additional new Hereditary Xerocytosis characteristics for HS and confirmed the heterogeneity of the Considered were 4 families with mutations in the PIEZO1 disease showing that changes in membrane conductance are not gene (Family 1 with patients P51.3 and P51.4; Family 2 with an overall feature of the disease but depend on the particular, P53.2 and P53.3; Family 3 with P50.2 and Family 4 with P52.1), specific mutation (Huisjes et al., unpublished). whose blood was delivered and recorded from, together with Interesting is that out of the many patients with SPTA1 the blood of a healthy subject (transportation control) (C51, mutations it is the two patients carrying a SPTA1 αLELY Frontiers in Physiology | www.frontiersin.org 7 April 2019 | Volume 10 | Article 386 Petkova-Kirova et al. Red Cell Electrophysiology in Anaemias FIGURE 5 | Whole-cell recordings of ion currents from RBCs of healthy donors and HS patients. Whole-cell recordings of ion currents from RBCs of healthy donors and HS patients with β-spectrin mutations (A,Ba,b). Compared are the I/V curves of P21.1 (n = 8) with its own transportation control C21 (n = 5) (Aa) as well as with a general control (n = 175) (Ab). Compared are the I/V curves of P10.1 (n = 3) with its own transportation control C10 (n = 6) (Ba) as well as with a general control (n = 175) (Bb). (Bc) Compares the I/V curve of the transportation control of P10.1, C10 (n = 6) with a general control (n = 175), where n denotes the number of cells. Currents were elicited by voltage steps from –100 to 100 mV for 500 ms in 20 mV increments at V h = –30 mV. Data are expressed as mean current density ± SEMs. Significant differences are determined based on an unpaired t-test with ∗ representing p < 0.05. Mutations below patients numbers are designated as amino acid substitutions in the respective protein. allele that show a change in their membrane conductance: own transportation control is also increased. Evident are an increase in their inward current. This holds true the large variations within the cells of the patients for for P18.1 vs. its own transportation control and vs. the both patient P18.1 and P19.1 but especially for P19.1. This general control of 175 cells and also for patient P19.1 might explain why the difference in the inward current vs. the pooled control. Although not reaching statistical between P19.1 and its transportation control does not reach a significance the inward current of P19.1 compared to its statistical significance. Frontiers in Physiology | www.frontiersin.org 8 April 2019 | Volume 10 | Article 386 Petkova-Kirova et al. Red Cell Electrophysiology in Anaemias FIGURE 6 | Whole-cell recordings of ion currents from RBCs of healthy donors and patients, carriers of several mutations and a complex genotype. Compared are the I/V curves of P11.1 (n = 11) with its own transportation control C11 (n = 13) (Aa) as well as with a general control (n = 175) (Ab). Compared are the I/V curves of P22.1 (n = 8) with its own transportation control C22 (n = 5) (Ba) as well as with a general control (n = 175) (Bb), where n denotes the number of cells. Currents were elicited by voltage steps from –100 to 100 mV for 500 ms in 20 mV increments at V h = –30 mV. Data are expressed as mean current density ± SEMs. Significant differences are determined based on an unpaired t-test with ∗ representing p < 0.05. Mutations below patients numbers are designated as amino acid substitutions in the respective proteins. Additionally to having an α-spectrin mutation P22.1 is PK deficiency carrier as well as an α-thalassemia carrier. Allele αLELY is a common polymorphic allele and its presence P22.1 with a mutation in α-spectrin as well as being a pyruvate in humans is by itself asymptomatic. The αLELY allele, however, kinase deficiency and a thalassemia carrier show an increase plays the role of an exacerbating factor when it occurs in both in their inward and outward current. While in the case trans to an α-spectrin mutation resulting in a disastrously of P22.1 such an increase cannot be straightforwardly explained weak spectrin network (Viel and Branton, 1996; Iolascon et al., as neither thalassemia nor pyruvate kinase deficiency alone give 2003). This is because, due to their reduced ability to form any change in conductance, P11.1 is particularly interesting. dimers, α chains from αLELY alleles are underrepresented in The Rh-associated glycoprotein (RhAG) coded by the RHAG the mature RBC cytoskeleton. Accordingly underrepresented are gene, together with the RhD and RhCcEe proteins, is a major any spectrin mutations found on the same allele and in turn component of the Rh blood group system. It is essential for overrepresented if found on the opposite allele (Wilmotte et al., assembly of the Rh protein complex in the RBC membrane and 1997). How a destabilized cytoskeleton might have an effect for expression of the Rh antigens (Avent and Reid, 2000). The on membrane conductance is a subject of speculations but as exact function of RhAG is not completely understood but it is the RBC membrane has little structural integrity without the suggested to be involved in RBC gas exchange as it promotes support of an intact and steady protein scaffold below, it might transmembrane NH3 transport (additionally NH4 + ) (Bakouh be that conformational changes influence the proper functioning et al., 2006) as well as facilitates CO2 membrane permeation of channels and lead to increased membrane conductance. (Endeward et al., 2008). More interesting is, however, that RhAG Noteworthy, in many cases, aggravating conditions such as can act as a pore for monovalent cations (Na+ , K+ , and Li+ ) an αLELY allele or a superimposed erythrocytic defect lead to an and RHAG point mutations leading to Ile61Arg and Phe65Ser enhanced membrane conductance and a leaky cell. Thus P11.1 substitutions result in massively increased permeabilities for K+ with a mutation in β-spectrin as well as a mutation in RHAG and and Na+ and to overhydrated stomatocytosis (Bruce et al., 2009). Frontiers in Physiology | www.frontiersin.org 9 April 2019 | Volume 10 | Article 386 Petkova-Kirova et al. Red Cell Electrophysiology in Anaemias FIGURE 7 | Whole-cell recordings of ion currents from RBCs of healthy donors and HX patients. Compared are the I/V-curves of all HX patients (N = 6) with the I/V curves of their own transportation controls (N = 4) (Aa) and with the I/V curves of all healthy subjects delivered throughout the study (N = 27) (Ab), where N denotes the number of healthy subjects or HX patients. No changes were observed in capacitance either with the transportation (0.725 pA/pF patients vs. 0.71 pA/pF transportation control; p > 0.05) or with the general control (0.725 pA/pF patients vs. 0.69 pA/pF general control; p > 0.05). Compared are the I/V curves of P51.3 and P51.4 pooled together (Family 1) (n = 22) with their own transportation control C51 (n = 7) (Ba) and the I/V curves of P53.2 and P53.3 pooled together (Family 2) (n = 27) with their own transportation control C53 (n = 9) (Ca), where n denotes the number of cells from the patients or the controls. (Bb,Cb) Compare the I/V curve of a general control based on currents recorded from all cells of all healthy subjects delivered throughout the study (n = 175) with the I/V curve of P51.3 and P51.4 pooled together (n = 22) (Bb) and P53.2 and P53.3 pooled together (n = 27) (Cb) (n denotes number of cells). P51.3 and P51.4 pooled together (Family 1) and P53.2 and P53.3 pooled together (Family 2) did not show a difference in their capacitance compared to the general or transportation control (0.69 pA/pF P51.3 and P51.4 vs. 0.72 pA/pF C51, p > 0.05; 0.69 pA/pF P51.3 and P51.4 vs. 0.69 pA/pF general control, p > 0.05; 0.75 pA/pF P53.2 and P53.3 vs. 0.71 pA/pF P53, p > 0.05; 0.75 pA/pF P53.2 and P53.3 vs. 0.69 pA/pF general control, p > 0.05) Currents were elicited by voltage steps from –100 to 100 mV for 500 ms in 20 mV increments at V h = –30 mV. Data are expressed as mean current density ± SEMs. Significant differences are determined based on an unpaired t-test with ∗ representing p < 0.05. Mutations below patients numbers are designated as amino acid substitutions in the respective protein. Expression of the mutated RhAGs in xenopus oocytes confirms substitution Val270Ile residing in the 5th endoloop of RhAG the large monovalent leaks imposed by the mutations and (Huang et al., 1999) although not linked to overhydrated modeling studies correlate those leaks with possibly widening stomatocytosis relates to the Rhnull syndrome accordingly the pore structures permitting passive diffusion of Na+ and characterized by varying degrees of chronic haemolytic anemia K+ (Bruce et al., 2009). Mutation c.808G > A in P11.1 with a and spherostomatocytosis (Nash and Shojania, 1987). Without Frontiers in Physiology | www.frontiersin.org 10 April 2019 | Volume 10 | Article 386 Petkova-Kirova et al. Red Cell Electrophysiology in Anaemias FIGURE 8 | Whole-cell recordings of ion currents from RBCs of healthy donors and HX patients. Compared are the I/V curves of P50.2 (n = 14) with its own transportation control C50 (n = 7) (Aa) as well as with a general control (n = 175) (Ab). Comparison of capacitance of P50.2 with control capacitances gave no difference with the general or the transportation control (0.73 pA/pF P50.2 vs. 0.80 pA/pF transportation control; p > 0.05; 0.73 pA/pF P50.2 vs. 0.69 pA/pF general control; p > 0.05). Compared are the I/V curves of P52.1 (n = 20) with its own transportation control C52 (n = 11) (Ba) as well as with a general control (n = 175) (Bb), where n denotes the number of cells. No changes were observed in capacitance either with the transportation (0.75 pA/pF P52.1 vs. 0.62 pA/pF C52; p > 0.05) or with the general control (0.75 pA/pF P52.1 vs. 0.69 pA/pF general control; p > 0.05). Currents were elicited by voltage steps from –100 to 100 mV for 500 ms in 20 mV increments at V h = –30 mV. Data are expressed as mean current density ± SEMs. Significant differences are determined based on an unpaired t-test with ∗ representing p < 0.05. Mutations below patients numbers are designated as amino acid substitutions in the respective protein. knowing the mechanism that might relate mutation RHAG current and, although counterintuitive, also accompanied by a c.808G > A (or the complex defect c.808G > A+SPTB significant loss of K+ from the RBC. c.154delC) to changes in the RBC membrane conductance, The major function of band 3 protein is of an anion P11.1 has been described with having less K+ in the transporter exchanging a bicarbonate for a chloride ion across plasma than its transportation control yet more than a non- the RBC plasma membrane thus ensuring efficient removal of transported normal control and a much increased activity CO2 from tissues (Guyton and Hall, 2006). However, data in of its Na+ /K+ pump (Huisjes et al., unpublished). Thus, it the literature show that point mutations resulting in single could well be that a possibly increased K+ leak underlined amino acid substitutions cause Na+ and K+ leaks with anion by the detected increased membrane conductance triggers transport activity either maintained or abolished depending compensatory changes, namely enhanced ion pumping that on the mutation (Bruce et al., 1993, 2005; Salhany et al., might explain the partially compensated K+ leak (i.e., that K+ 1995; Stewart et al., 2010, 2011). Those mutations causing in the plasma of the patient is less than K+ in the plasma predominantly stomatocytosis but also spherocytosis (Arakawa of its transportation control). This is in line with observations et al., 2015) have been suggested to induce monovalent cation that haemolytic diseases showing increased non-Na+ /K+ pump leaks in one of 3 possible ways: (i) converting the anion and non-NaK2Cl cotransport, K+ fluxes [ouabain- (an inhibitor exchanger in a non-selective cation conductor, (ii) inducing of the Na+ /K+ pump) and bumetanide-(an inhibitor of the cation conductance in a still functioning exchanger and (iii) NaK2Cl cotransport) resistant K+ fluxes] are accompanied causing the anion exchanger to stimulate endogenous cation by increased Na+ /K+ pump fluxes (ouabain- sensitive fluxes) transporters (Badens and Guizouarn, 2016). The latter has (Stewart, 2004). been shown in heterologous expression systems (Stewart et al., Not always, however, a resulting changed membrane 2010) as well as in RBCs (H734R) (Bogdanova et al., 2009). conductance is manifested as an increase in current. A patient It could indeed be that band 3 protein is engaged in complex (P12.1) with a mutation in band 3 protein shows a decrease in interactions modulating cation permeability pathways (both Frontiers in Physiology | www.frontiersin.org 11 April 2019 | Volume 10 | Article 386 Petkova-Kirova et al. Red Cell Electrophysiology in Anaemias FIGURE 9 | Whole-cell recordings of ion currents from RBCs of healthy donors and patients with enzymopathies (glucose-6-phosphate dehydrogenase deficiency and pyruvate kinase deficiency). Compared are the I/V-curve of all glucose-6-phosphate dehydrogenase deficiency patients (N = 3) with the I/V curve of their own transportation controls (N = 3) (Aa) and with the I/V curve of all healthy subjects delivered throughout the study (N = 27) (Ab), where N denotes the number of healthy subjects or patients. Capacitance of the three patients P40.1, P41.1, P42.1 was not any different from that of the controls (0.62 pA/pF P40.1 + P41.1 + P42.1 vs. 0.68 pA/pF C40 + C41 + C42, p > 0.05; 0.62 pA/pF P40.1 + P41.1 + P42.1 vs. 0.69 pA/pF general control, p > 0.05). Compared are the I/V-curve of all glucose-6-phosphate dehydrogenase deficiency patients cells (n = 27) with the I/V curve of all transportation control cells (n = 22) (Ba) as well as with a general control (n = 175) (Bb), where n denotes the number of cells. Compared are the I/V curves of P45.1 (“PK” below the patient number stands for pyruvate kinase deficiency) (n = 15) with its own transportation control C45 (n = 5) (Ca) as well as with a general control (n = 175) (Cb). No changes were observed in capacitance either with the transportation (0.72 pA/pF P45.1 vs. 0.68 pA/pF C45; p > 0.05) or with the general control (0.72 pA/pF P45.1 vs. 0.69 pA/pF general control; p > 0.05). Currents were elicited by voltage steps from –100 to 100 mV for 500 ms in 20 mV increments at V h = –30 mV. Data are expressed as mean current density ± SEMs. channels and transporters) and that mutations changing its between band 3 protein and TRPC6 in erythrocytes as well. conformation or its availability in the membrane might lead to While no changes in cation channel activity (not an increase multifaceted effects either increasing or decreasing membrane either, regardless of the reported substantially elevated cation conductance (both of which with the detrimental result of leak) have been detected in band 3 protein R730C RBCs disturbing RBC ion homeostasis). In line is a study showing [as measured by on-cell patch-clamp, (Stewart et al., 2011)] kidney band 3 protein interaction with nephrin (Wu et al., as well as in band 3 protein H734R RBCs [as judged by 2010). The intracellular domain of nephrin interacts with membrane potential changes, (Bogdanova et al., 2009)], it could TRPC6, believed to be present in erythrocytes (Foller et al., 2008; be that each mutation alters in a different way endogenous Danielczok J. et al., 2017) suggesting a possible functional link permeability pathways. Frontiers in Physiology | www.frontiersin.org 12 April 2019 | Volume 10 | Article 386 Petkova-Kirova et al. Red Cell Electrophysiology in Anaemias FIGURE 10 | Whole-cell recordings of ion currents from RBCs of healthy donors and patients with enzymopathies. Compared are the I/V curves of P43.1 (GCLD below the patient number stands for glutamate-cysteine ligase deficiency) (n = 6) with its own transportation control C43 (n = 5) (Aa) as well as with a general control (n = 175) (Ab). No changes were observed in capacitance either with the transportation (0.64 pA/pF P43.1 vs. 0.65 pA/pF C43; p > 0.05) or with the general control (0.64 pA/pF P43.1 vs. 0.69 pA/pF general control; p > 0.05). Compared are the I/V curves of P44.1 (GRD below the patient number stands for glutathione reductase deficiency) (n = 9) with its own transportation control C44 (n = 5) (Ba) as well as with a general control (n = 175) (Bb), where n denotes the number of cells. No changes were observed in capacitance either with the transportation (0.76 pA/pF P44.1 vs. 0.65pA/pF C44; p > 0.05) or with the general control (0.76 pA/pF P44.1 vs. 0.69 pA/pF general control; p > 0.05). Currents were elicited by voltage steps from −100 to 100 mV for 500 ms in 20 mV increments at V h = −30 mV. Data are expressed as mean current density ± SEMs. FIGURE 11 | Whole-cell recordings of ion currents from RBCs of healthy donors and a patient with β-thalassemia. Compared are the I/V curves of P60.1 (n = 12) with its own transportation control C60 (n = 5) (A) as well as with a general control (n = 175) (B). No changes were observed in capacitance either with the transportation (0.72 pA/pF P60.1 vs. 0.64 pA/pF C60; p > 0.05) or with the general control (0.72 pA/pF P60.1 vs. 0.69 pA/pF general control; p > 0.05). Currents were elicited by voltage steps from –100 to 100 mV for 500 ms in 20 mV increments at V h = –30 mV. Data are expressed as mean current density ± SEMs. Frontiers in Physiology | www.frontiersin.org 13 April 2019 | Volume 10 | Article 386 Petkova-Kirova et al. Red Cell Electrophysiology in Anaemias Regarding mutation Ile783Asn of patient P12.1, it is not in healthy subject is using the blood of several healthy subjects as a the cytoplasmic half of the core domain of band 3 protein, transportation control or, even better, the blood of several healthy where most of the mutations causing stomatocytosis are relatives. A further problem, however, is that transportation (Arakawa et al., 2015), yet it is very close to and in the same could have different effects on the patient and on the control. transmembrane domain (TM 12) as another mutation, namely This problem could be avoided by avoiding transportation itself, Gly796Arg, also triggering stomatocytosis (Arakawa et al., 2015). whenever possible. According to (Huisjes et al., unpublished) mutation Ile783Asn is accompanied by increased Na+ -K+ -ATPase activity, a referral Hereditary Xerocytosis to stomatocytosis, yet by an osmoscan curve with a typical HS Piezo 1 is a mechanically activated cation channel (Coste pattern and extremely low eosin-5-maleimide (EMA) staining et al., 2010, 2012), which is permeable to monovalent cations (61%), likely reflecting strongly reduced copy numbers of band (PK > PCs ∼ = PNa > PLi ) and to most divalent cations like 3 protein, a referral to spherocytosis. (EMA-binding on RBCs Ba2+ , Ca2+ , and Mg2+ , but not Mn2+ (Gnanasambandam involves the ε-NH group of lysine at position 430 from band et al., 2015). Expressed in many tissues like kidney, lung and 3 protein (Nicolas et al., 2003) and is experimentally found to urinary bladder (Coste et al., 2010; Miyamoto et al., 2014), correlate with band 3 protein expression on RBCs (Huisjes et al., Piezo 1 has been detected in the plasma membrane of RBCs, 2018). Thus the question whether it is the unavailability of band e.g., by mass spectroscopy and immunologically (Zarychanski 3 protein or a possible structural and conformational change that et al., 2012; Andolfo et al., 2013; Kaestner and Egée, 2018). causes the decreased outward current, remains open. A major role of Piezo1 channels in RBCs is in volume In our study, as outlined in the Results section, we have regulation and mutations in the channel have been linked to compared the currents measured from the RBCs of our HX, a dominantly inherited haemolytic anemia, characterized patients once with their transportation control, i.e., currents by decreased K+ and increased Na+ RBCs content, as well as measured from the RBCs of a healthy subject, whose blood dehydration resulting in increased mean corpuscular hemoglobin was delivered together with the blood of the patient, and concentrations (MCHC), a leftward shift of the osmotic gradient once with a general control, i.e., currents measured from the ektacytometry curve and increased osmotic resistance of the cells of all healthy subjects delivered throughout the study. RBCs (Gallagher, 2013; Shmukler et al., 2014; Glogowska Inevitably a question comes up, especially when there are and Gallagher, 2015; Andolfo et al., 2016). Disease clinical differences in the comparisons with the two controls, which is manifestations are variable and may include mild to moderate the more appropriate one. Whereas, undoubtedly, considering haemolysis, perinatal edema and non-immune hydrops fetalis the transportation control, allows us to take into account that spontaneously resolve, thrombosis, pseudohyperkalemia and the particular transportation conditions such as temperature, sometimes severe iron overload in the course of the disease vibration intensity and shipment duration, it has limitations. (Gallagher, 2013; Glogowska and Gallagher, 2015; Andolfo et al., Such limitations are the low number of measured cells but mostly 2016). Out of the four mutations in our study [R2456H (P51.3 the fact that the control subject, although judged healthy, might and P51.4), R2088G (P53.3 and P53.2), C426R (P50.2), and not be a representative control. Thus in the case with C17, the E2496ELE (P52.1)], three (R2456H, R2088G, and E2496ELE) control for patients P17.1 and P17.2, the averaged capacitance are known and have been extensively characterized including of C17 RBCs (0.53 pA/pF) is statistically significantly smaller expression of the mutant Piezo1 channel in heterologous systems than the averaged capacitance of the RBCs of the general control and characterization of the channel activity (Zarychanski et al., (0.69 pA/pF). This in turn results in an increased current density 2012; Albuisson et al., 2013; Andolfo et al., 2013; Bae et al., (current divided by capacitance) for the transportation control 2013; Sandberg et al., 2014; Shmukler et al., 2014; Glogowska which might explain the observed difference of P17.1 and P17.2 et al., 2017). Two of the mutations, R2456H and E2496ELE, compared to C17 but not to the general control (Figures 4B,C). among the most common ones found in typical HX patients With C10, the control of P10.1, the situation is even more (Zarychanski et al., 2012; Albuisson et al., 2013; Andolfo et al., extreme, as, even though the averaged capacitance of the RBCs 2013; Shmukler et al., 2014), are in the C-terminal region, of the transportation control is higher compared to the averaged part of the pore module of Piezo1 (Coste et al., 2015; Ge capacitance of the general control, which results in a lower et al., 2015). Those mutations do not alter the sensitivity of current density, the current density still remains much higher the channel to mechanical stimulation but cause considerable than the one of the general control. The appearance of the I/V increase in the inactivation time constant thus giving rise to curve is also very different with the reversal potential being much an increased channel activity in response to a given mechanical shifted to the more negative values compared to the general stimulus (Albuisson et al., 2013; Glogowska et al., 2017). Could control (Figure 5Bc). The above mentioned limitations could an increased channel activity lasting only for the duration be avoided by considering the general control which, due to of a short mechanical stimulus explain the ion disbalance the high number of cells, is balancing (smoothing out) the observed in HX? A possible answer is that during their effect of a healthy but unrepresentative subject and is close circulation in the vascular system RBCs are under constant to an ‘ideal’ control. At the same time what is an advantage mechanical stress squeezing in capillaries and in the tiny slits of the general control is simultaneously a disadvantage as it of the spleen undergoing numerous rounds of mechanical ‘balances’ also the specific transportation effects on the samples. stimulation with Piezo1 activation (Danielczok J.G. et al., 2017) A way out of accidentally coming across a non-representative and, in the case of Piezo1 prolonged inactivation, not being Frontiers in Physiology | www.frontiersin.org 14 April 2019 | Volume 10 | Article 386 Petkova-Kirova et al. Red Cell Electrophysiology in Anaemias capable of replenishing their ions. However, it is also likely disorders, we stumble upon a great variability in results. Such that slowing of inactivation could potentially cause a slight a variability is of course reflecting the many triggers of the increase in a basal Piezo1 activity (independent of mechanical disbalance and for sure not lessened by the fact that not only stimulation) (Albuisson et al., 2013). This is supported by the between different mutations but also among members of a study of (Andolfo et al., 2013), which demonstrates spontaneous family with the same mutation there are distinct differences ion channel activity in R2456H patient RBCs, blocked by starting from the severity of the disease and extending down GsMTx-4 and not observed in healthy cells. In addition are to the cellular level. Such a summary is even harder and the on-cell patch recordings from the RBCs of another R2456H certainly not helped by the great variability in healthy subjects patient revealing once again an increased cation channel activity reflected in control membrane conductance measurements as (independent of mechanical stimulation) without information on seen in our study. And last but not least answers are yet the identity of the channel (Shmukler et al., 2014). Consistent heavier without so far clearly knowing how an increased with Piezo 1 increased activity having direct or secondary Na+ and a decreased K+ can lead to overhydration as in effects on membrane conductance is the observed decrease in OHSt or to dehydration as in HX. Nevertheless based on conductance in P52.1 (E2496ELE) RBCs. Also in P51.3 and our study we conclude that changes in conductance are P51.4 (R2456H) and P52.1 (E2496ELE) a rightward shift of incurred by certain α-spectrin [c.2755G > T (p.Glu919) and the I/V curve is observed (Figures 7Bb, 8Bb) indicative of an c.678G > A p.(Glu227fs) whenever an αLELY allele is present], upregulation/downregulation of a channel/channels. Whether band 3 protein [c.2348T > A p.(Ile783Asn)] and Piezo1 the shift is pertinent to the mutations though is not explicitly (c.7483_7488dupCTGGAG p.2495_2496dupLeuGlu) mutations clear as such a shift is not detected when patients’ currents are as a difference is observed with both the general and the compared with their transportation controls (Figures 7Ba, 8Ba). transportation control. Identification of the channel/channels Concerning mutation C426R (P50.2) which shows an increase that underlie the changed conductances demands future studies. in current only with the general control but not with its transportation control we tend to disregard the difference shown with the general control as the transportation control ETHICS STATEMENT was taken from a genetically related healthy subject and it indeed shows a remarkable similarity with the I/V curve of the Patients’ data were handled anonymously as outlined in the ethics patient (Figure 8Aa). agreements. These agreements were approved by the Medical Ethical Research Board (MERB) of the University Medical Center Utrecht, Netherlands (UMCU) under reference code 15/426M Enzymopathies and Beta-Thalassemia “Disturbed ion homeostasis in hereditary hemolytic anemia” and Metabolic enzyme deficiencies (glucose-6-phosphate by the Ethical Committee of Clinical Investigations of Hospital dehydrogenase deficiency, pyruvate kinase deficiency, glutamate- Clinic, Spain (IDIBAPS) under reference code 2013/8436. cysteine ligase deficiency and glutathione reductase deficiency) as well as β-thalassemia are not accompanied by changes in membrane conductance. The former is consistent with the lack of changes also in the K+ and Na+ content of 11 patients AUTHOR CONTRIBUTIONS with congenital non-spherocytic haemolytic anemia including All authors listed have made a substantial, direct and intellectual pyruvate kinase deficiency (Vives Corrons and Besson, 2001). contribution to the work, and approved it for publication. CONCLUSION FUNDING Trying to summarize and come up with a common channel The research leading to these results has received funding increased activity/dysfunction or just an effect (decrease or from the European Seventh Framework Program under grant increase in conductance) accompanying RBCs ion disbalance agreement number 602121 (CoMMiTMenT). REFERENCES Andolfo, I., Russo, R., Gambale, A., and Iolascon, A. (2016). New insights on hereditary erythrocyte membrane defects. Haematologica 101, 1284–1294. doi: Albuisson, J., Murthy, S. E., Bandell, M., Coste, B., Louis-Dit-Picard, H., 10.3324/haematol.2016.142463 Mathur, J., et al. 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The exon 46-encoded sequence is essential for stability distributed under the terms of the Creative Commons Attribution License (CC BY). of human erythroid alpha-spectrin and heterodimer formation. Blood 90, The use, distribution or reproduction in other forums is permitted, provided the 4188–4196. original author(s) and the copyright owner(s) are credited and that the original Wu, F., Saleem, M. A., Kampik, N. B., Satchwell, T. J., Williamson, R. C., Blattner, publication in this journal is cited, in accordance with accepted academic practice. S. M., et al. (2010). Anion exchanger 1 interacts with nephrin in podocytes. No use, distribution or reproduction is permitted which does not comply with J. Am. Soc. Nephrol. 21, 1456–1467. doi: 10.1681/ASN.2009090921 these terms. Frontiers in Physiology | www.frontiersin.org 17 April 2019 | Volume 10 | Article 386