Ca²⁺ increase in RBCs with Gárdos channel mutations. (A) Ca²⁺ increase in RBCs of a patient with the p.S314P mutation (AIII.1 in Fermo et al¹³) in comparison with healthy control RBCs. (a-b) Representative fluorescence images of RBCs stained with Fluo-4; scale bar, 10 μm. (c) Statistical analysis of fluorescent images as depicted in subpanel a. The white numbers refer to the numbers of analyzed cells. (d) Analysis of flow cytometric measurements of Fluo-4–stained RBCs. Statistical differences were checked with a Student t test; *** P < .001. (B) Mechanistic hypothesis regarding how Gárdos channel activity may trigger an increase in intracellular RBC Ca²⁺ concentration. The membrane potential changes induced by the opening of the Gárdos channel (cp. explanation in "Results and discussion") activates the voltage-gated Ca²⁺ channel Ca_V2.1, possibly modulated by the mechanosensitive Piezo1 channel. If Piezo1 is blocked, the Gárdos channel–induced opening of Ca_V2.1 would be maximal. In contrast, Piezo1 activity, due to the nonselective cation permeability of the channel, would “dissipate” the Gárdos channel–induced hyperpolarization. As a consequence, Ca_V2.1 activity would be reduced. The scheme contains annotations for the experimental manipulations of the molecular players. An arrow refers to activation and an anchor to inhibition of the transport protein.