Reduced Mg²⁺ in CF cells impairs GTP activation of Rab27a. (A) Maximum increase in GTP-bound Rab27a following TNF-α treatment was compared between HC and ΔF508/ΔF508 CF (CFΔ) neutrophils and between vehicle control treated (0.05% v/v DMSO; VC) or CFTRinh-172 (10 μM; Inh) treated HC cells (CFΔ and Inh expressed relative to HC and VC, respectively). Reduction in maximum Rab27a activity in inhibitor-treated HC cells is similar to CFΔ cells (P = .03 between HC and CFΔ, P = .01 between VC and Inh, and P = .2 between CFΔ and Inh, Student t test, n = 3 subjects per group). (B) The cytosolic Mg²⁺ concentration in HC cells was incrementally decreased from 0.8 to 0.5 mM using the ionophore A23187 (20 μM), and maximum increase in GTP-bound Rab27a following TNF-α treatment was analyzed and expressed relative to 0.8 mM (P = .0001 between 0.5 and 0.6 mM, P = .005 between 0.6 and 0.7 mM, and P = .008 between 0.7 and 0.8 mM, Student t test, P = .0001 by 1-way ANOVA, n = 3 subjects per group). (C) Cytosolic GTP content of HC neutrophils or cells from patients with CF of the ΔF508/ΔF508 (CFΔ) or G551D/ΔF508 (CFG) genotype was equal as measured by high-performance liquid chromotography (P = .9, 1-way ANOVA, n = 3 subjects per group). (D) Serum Mg²⁺ levels of HC and PWCF were not statistically different (P = .6, Student t test, n = 28 subjects per group) and were within the normal range (hashed lines). All measurements are means ± SEM from biological replicates.