Figure 4. ATP2B4 activity and calcium homeostasis in erythroid cells.
Ratiometric Fura-Red fluorescence (A) prior to and (B) following application of caffeine (10 μM) in HUDEP-2 WT (basal, n = 21; caffeine treated, n = 5), nondeleted (nondeletion: basal, n = 25; caffeine treated, n = 14), and ATP2B4 enhancer–deleted cells (biallelic deletion: basal, n = 30; caffeine treated, n = 27). Box plots represent the median (central line), the first and third quartiles (hinges), and the lowest and highest values inside 1.5 times the interquartile range from the hinges (whiskers). Statistical analyses used Welch’s t test. Two-tailed P values are reported. **P < 0.01; ****P < 0.0001. (C) Representative time course of the fluorescence Fura-Red ratio following exposure to a single bolus of caffeine (black arrow) in a control cell (black, WT) and in a cell with biallelic deletion of the ATP2B4 enhancer (red; biallelic deletion). Intracellular calcium store release induced by caffeine exposure substantially increased cytoplasmic levels in both cells, but elevated calcium concentrations persisted only in cells with the ATP2B4 enhancer deleted. (D) Model of genetic variation at the ATP2B4 erythroid enhancer influencing rbc traits and malaria susceptibility. Erythroid cells carrying the minor haplotype at the erythroid-specific enhancer express lower levels of ATP2B4. The rbc with reduced ATP2B4 accumulate cytoplasmic calcium, resulting in dehydration and elevated MCHC. Although relatively resistant to infection by the malaria parasite P. falciparum, dehydrated rbc may increase the severity of erythroid disorders such as sickle cell disease (SCD).