Erythrocytes from hereditary xerocytosis patients heterozygous for KCNN4 V282M exhibit increased spontaneous Gardos channel‐like activity inhibited by senicapoc
Authors: Alicia Rivera, David H. Vandorpe, Boris E. Shmukler, Denis R. Gallagher, Christopher C. Fikry, Frans A. Kuypers, Carlo Brugnara, L. Michael Snyder, Seth L. Alper
Journal: American Journal of Hematology (2017)

Hereditary Xerocytosis (HX) is an autosomal dominant hemolytic anemia of variable penetrance associated with red cell dehydration, elevated corpuscular hemoglobin concentration mean (CHCM) and resistance to hypo-osmotic lysis. These properties are accompanied less reliably by macrocytosis and stomatocytosis of low-to-moderate frequency. The disease prevalence originally estimated at 1 per 10,000 may be considerably higher.1 Most HX is caused by heterozygous missense mutations in PIEZO1, a mechanosensitive, Ca2+-permeable cation channel polypeptide of 2521 aa in length.2 The mutations confer gain-of-function arising from delayed inactivation of the cation channel,3 leading to increased Ca2+ entry into the red cell.4 Prolongation of the brief elevations in HX red cell cytosolic Ca2+ that follow transient openings of mutant PIEZO1 channels are believed to increase activation of the erythroid Gardos channel, KCNN4, leading to red cell dehydration and MCHC elevation.4 Indeed, pathogenic gain-of-function KCNN4 mutations R352 (in the calmodulin-binding domain), V282M, and V282E (at the cytoplasmic face of TM6) have been reported in families with normal PIEZO-1.5-7 The central role of KCNN4 activity in the pathogenesis of HX has encouraged the proposal that the KCNN4 antagonist senicapoc should be clinically tested in HX. Susceptibility to inhibition by the KCNN4 antagonist, senicapoc, is preserved by some HX-associated KCNN4 mutants, as evidenced by whole cell recording of recombinant channel activity expressed in HEK-293 cells. KCNN4 V282M retained native senicapoc affinity, whereas senicapoc inhibition of KCNN4 R352H was 10-fold more potent and KCNN4 V282E exhibited severely reduced affinity.8 Here, we report our results characterizing red cell KCNN4 function and senicapoc sensitivity and other red cell properties in a cohort of HX patients heterozygous for KCNN4 mutation V282M (family WO in reference 5's Figure 2B).5 HX patients heterozygous for KCNN4 V282M were anemic, with hematocrit of 35.2 ± 0.9% (n = 22) vs control value of 45 ± 1.0% (n = 21; P < 10−5), and with reticulocytosis of 9.9± 1.0% (n = 22) vs. control value of 1.4 ± 0.1% (n = 21). HX red cells from patients heterozygous for KCNN4 V282M also exhibited a severely left-shifted osmotic fragility curve (Figure 1A), indicative of extreme resistance to osmotic lysis. This resistance to osmotic lysis corresponded to a substantial right-shift in the phthalate density profile (Figure 1B), demonstrating substantially increased density of HX red cells. HX red cells also showed reduced K content (178 ± 32 vs. 314 ± 45 mmol/kg Hb), with increased Na content (41 ± 5 vs 24 ± 5 mmol/kg Hb; n = 12, P < 10−6 for both). Osmotic deformability and fragility, density, and basal KCNN4-like activity of HX RBC from patients heterozygous for KCNN4 mutation V282M. A. Osmotic fragility index (OFI) was measured in freshly isolated blood from two V282M HX patients (IV.5 and IV.10) heterozygous for KCNN4 V282M (red squares, OFI = 92 ± 7) and from two normal subjects (black diamonds, OFI = 154 ± 5). Means ± SEM (n = 4, P = .00002). B. Phthalate density profiles indicated D50 values of 1.117 ± 0.0 for RBC from V282M HX patients IV.10 and III.3 (red circles) and 1.096 ± 0.002 from two normal controls (black diamonds). Means ± SD (n = 3, P < .0003). C. Osmotic ektacytometry performed with overnight-shipped blood yielded values for Omin (osmolality at which 50% of cells hemolyze), DImax (maximal cell deformability), and Ohyp (osmolality at which DImax is half maximal). RBC from HX patients III.5 (red) and IV.5 (green trace; see pedigree of Figure 2B in Andolfo et al.5) were compared with shipment-control RBC (blue trace) and fresh local donor RBC (gray and black traces). Black squares show range (±SD) for control RBC. D. A representative on-cell patch clamp gigaseal recording9 from a single V282M HX RBC (–Vp = +50 mV) before (upper current trace, -SCP) and after superfusion of 200 nM senicapoc (lower trace, +SCP). Left panel i: NPo values for on-cell patch currents recorded from normal control (V282, n = 3 RBC) and HX RBC (V282M; n = 6 RBC from patients IV:5 and IV:10, means ± SEM; *, P = .02). Right panel ii: NPo values for currents (n = 3) recorded from HX cells before (V282M) and during superfusion of 200 nM senicapoc (V282M + SCP; *, P = .02). Symmetric pipette and bath solutions contained (in mM) 140 NaCl, 5 KCl, 1 CaCl2, 1 MgCl2, 10 Na HEPES, pH 7.40. E. Net K+ efflux from wild type (V282) and from HX RBC (V282M) measured at 37°C in the presence of ouabain (0.1 mM) and bumetanide (1 mM) as previously described,10 in the absence (white bars) or presence (black bars) of 200 nM senicapoc (SCP). Means ± SEM from 4 triplicate experiments using blood from 3 normal controls and from patients III.5, IV.5, and IV.10. **, P < .001 for V282 vs V282M (white bars); *, P < .05 for V282M vs V282M + SCP; NS, not significant. F. Unidirectional K+ influx into normal control (V282) and HX RBC (V282M) at 37°C in the presence of ouabain (0.1 mM) and bumetanide (1 mM), measured as previously described10 using 86Rb+ influx, in the absence (white bars) or presence (black bars) of 200 nM senicapoc (SCP). Means ± SEM from 4 triplicate experiments using blood from 3 normal controls and from patients III.5, IV.5, and IV.10. **, P < .04 for V282 WT vs V282M (white bars); *, P < .05 for V282M vs V282M + SCP The ektacytometry profile of red cells heterozygous for KCNN4 V282M is left-shifted to an unusually extreme degree, with reductions in Dmax as well as of Ohyp values (Figure 1C; left shift is too extreme to allow determination of Omin value). This left-shifted curve indicating reduced shear-sensitive deformability is consistent with profound red cell dehydration and contrasts with the relatively normal ektacytometry profiles reported for HX red cells from patients heterozygous for KCNN4 R352H.5 Whereas control red cells exhibited minimal channel activity in on-cell patch recordings, red cells from patients heterozygous for KCNN4 V282M showed increased spontaneous channel activity. NPo was ∼eightfold elevated and inhibited nearly completely by 200 nM senicapoc (Figure 1D). This increased basal channel activity (unitary chord conductance ∼12 pS, not shown) was reflected in increased net K+ efflux insensitive to ouabain plus bumetanide, but partially sensitive to 200 nM senicapoc (Figure 1E), a component of activity undetected in control red cells. Moreover, unidirectional K+ influx (measured as 86Rb+) insensitive to ouabain plus bumetanide but sensitive to senicapoc was undetected in unstimulated control red cells, but present in unstimulated HX red cells from patients heterozygous for KCNN4 V282M (Figure 1F). The combined data indicate that HX red cells from anemic patients heterozygous for KCNN4 mutation V282M exhibit severe dehydration characterized by high red cell density with resistance to deformability and to osmotic lysis. Red cells from patients heterozygous for the KCNN4 mutation V282M exhibit increased basal senicapoc-sensitive channel activity, accompanied by increased senicapoc-sensitive net K+ efflux and increased senicapoc-sensitive unidirectional K+ influx. The demonstration by several methods of increased senicapoc-sensitive, basal K+ leak from HX red cells of patients heterozygous for the causative KCNN4 mutation V282M strengthens the proposal of senicapoc as a promising treatment for patients with this subtype of HX, as well as for HX secondary to the KCNN4 R352H mutation.5, 8 The relationship between HX red cell dehydration and the anemia of HX secondary to gain-of-function KCNN4 mutations remains to be investigated. Successful senicapoc treatment of anemia in HX patients would support the intrinsic pathogenicity of increased red cell density in this context, and perhaps more generally. We are indebted to our experimental subjects for providing blood samples. SLA was supported by funding from QUEST Diagnostics, Inc. FK was supported by CHORI. LMS, DRG, and CCF are employees of QUEST Diagnostics. CB is a consultant to Pfizer on sickle cell disease research. CB and SLA hold patents on the use of senicapoc for treatment of sickle cell disease and other diseases with red cell dehydration.