Functional tests of the new Gardos channel mutation p.S314P and relation to the mutation p.R325H. (A) Percoll density centrifugation of a blood sample from patient AIII.1 compared with a control subject. A methodologic description is given in the supplemental Material. (Ai) Image of the centrifuged tubes. (Aii) Profile of the cell distribution derived from intensity-based analysis of the image shown in panel Aa. Distance zero refers to light cells on top of the tube, and distance 2500 refers to the lowest measurable position in the tube. Low-density (L), medium-density (M), and high-density (H) fractions are marked with green, dark blue, and red bars, respectively. (B) Membrane potential measurements in RBC suspensions using the carbonyl cyanide-m-chlorophenylhydrazone method reported by Macey et al.¹⁵  A detailed description is provided in the supplemental Material. (Bi) Within approximately 3 minutes, RBCs reach an equilibrium at the RBC resting membrane potential of −10 mV. When the Ca²⁺ transporter A23187 at a concentration of 10 µM in the presence of 4 μM Ca²⁺ is added (equivalent to full Gardos channel activation), membrane potential drops immediately to approximately −80 mV due to the induced K⁺-conductance. Repolarization is accelerated when 10 µM of the Gardos channel inhibitor TRAM34 is added. Upon addition of TRITON X-100, which lyses the RBCs, a membrane potential no longer exists (0 mV). Here, mutated cells (KCNN4 p.S314P) react the same as healthy RBCs. (Bii) Within approximately 3 minutes, added RBCs reach an equilibrium at the RBC resting membrane potential of −10 mV. When 100 µM of the Gardos channel activator NS309 is added, the membrane potential of the healthy RBCs drops to approximately −15 mV, whereas in the mutated cells, the membrane potential reaches almost −50 mV (p.S314P) and −70 mV (p.R352H), hinting to gain-of-function mutations in both variants. At the end, TRITON X-100 is added to get the 0 mV calibration. Panels Biii and Biv are experiments performed in analogy to panel Bb but with the addition of 50 µM and 10 µM NS309, respectively. Blue lines represent normal control, green lines p.S414P mutated cells, and red lines p.R352H KCNN4 variant. (C) Whole-cell recordings of the Gardos channel currents from RBCs of a healthy subject and patient AIII.1 (p.S314P). A detailed methodologic description is presented in the supplemental Material. Currents were elicited by voltage steps from −130 mV to 50 mV for 500 ms in 20 mV increments at Vh = −30 mV and recorded in the absence and after application of 1 μM TRAM34, a specific Gardos channel blocker. (Ci) Raw current traces from a healthy subject’s RBCs in the absence (top left) and in the presence (bottom left) of 1 μM TRAM34 as indicated above the recordings. For comparison, raw current traces from the patient’s RBCs in the absence (top right) and in the presence (bottom, right) of 1 μM TRAM34 as indicated above the recordings. Note that in this particular example, the background current in the healthy control subject is higher than in patient AIII.1. (Cii) Comparison of the percent block by 1 μM TRAM34 of the mean currents at −110 mV in the healthy and mutated RBCs. Significance was checked based on an unpaired Student t test, *P < .05. (D) Correlation of TRAM34-induced change in current variability vs NS309-induced membrane potential change for healthy controls and cells with mutations p.S314P and p.R352H. The statistical values are patient based. Values of p.S314P variant are taken from panels B and C. Patch clamp recordings of p.R352H are from Fermo et al¹⁰  (n₁ = 23 cells) and 2 additional patients (n₂ = 9 cells; n₃ = 7 cells). NS309-induced potential changes were measured in three p.R325H patients (exemplified in panel Ca).