Experimental evidence for a mechanistic explanation. (A) Investigation of healthy RBCs to examine interactions between the Gárdos channel and other RBC ion channels. (a) Representative histogram from a flow cytometry measurement of RBCs stained with Fluo-4 and stimulated with 100 μM NS309. The red and the blue lines mark the populations of nonresponding and responding RBCs, respectively. (b) Number of RBCs responding with a high Ca²⁺ content as outlined in subpanel a at a concentration of 100 μM NS309 (left column) and the effect (preincubation) of the Gárdos channel inhibitors CTX (0.1 μM) and TRAM34 (1 μM) on NS309 (100 μM)-stimulated RBCs (right columns). (c) A new set of experiments compares the action (preincubation) of ω-Agatoxin TK (1 μM), a specific inhibitor of Ca_V2.1, and GsMTx-4 (2 μM), a toxin inhibiting the mechanosensitive channel Piezo1, on NS309 (100 μM)-stimulated RBCs. (b-c) All measurements are performed on at least 4 different donors. Plotted are mean values and the standard error of mean. Statistical differences were checked with a 1-way analysis of variance (ANOVA) and the Tukey multiple comparisons test. **** P < .0001; ns, not significant (P > .05). (d) Monitoring the membrane potential in a population of RBCs by the MBE method. The blue curve starts at the resting membrane potential (−12 mV). Addition of 15 μM A23187 leads to Ca²⁺ entry and the consecutive full activation of the Gárdos channel resulting in a hyperpolarization of −60 mV. Addition of TRAM34 inhibits the Gárdos channel resulting in a depolarization. The red curve also starts with the resting membrane potential and addition of 50 μM NS309 leads to the activation of the Gárdos channel, also resulting in a hyperpolarization but to a lesser extent as for the blue curve. Because we monitor the membrane potential in a cell population of >10⁸ RBCs, it is impossible to see a membrane potential flickering (compare "Results and discussion") that may happen in individual cells. The hyperpolarization to only −40 mV is therefore caused by, to a lesser extent, Gárdos channel activation, that is, a composition of cells with open and closed Gárdos channels. For both experiments, Triton-X 100 is used to calibrate for 0 mV. The curves present the mean of a triplicate measurement of a healthy donor and are a representative of 3 different donors. (e) Membrane potential upon 100 μM NS309 stimulation in dependence of the extracellular Ca²⁺ concentration. (f) Percentage of high Ca²⁺ RBCs upon 100 μM NS309 stimulation in dependence of the external Ca²⁺concentration. The corresponding histograms are provided in supplemental Figure 2. The bell-shaped curve can be explained by the Gárdos channel–Ca_V2.1 interaction (see main text). (e-f) All measurements were performed on 3 different donors. Plotted are mean values and the standard error of mean. (B) Testing the principle investigated in panel A on RBCs of patients carrying a Gárdos channel mutation. (a) Differential response (number of cells responding with increased intracellular Ca²⁺) of healthy and KCNN4 p.S314P- and p.R352H-mutated RBCs on stimulation with 10 μM NS309. (b) p.R352H-mutated RBCs were incubated for 24 hours with the Gárdos channel inhibitors Senicapoc (5 μM) and TRAM34 (1 μM), the Ca_V2.1 inhibitor ω-Agatoxin TK (1 μM), and the Piezo1 inhibitor GsMTx-4 (2 μM). The number of cells responding with increased intracellular Ca²⁺ is shown.