Figure 1.

NMDA receptor-mediated Ca²⁺ entry in human neutrophils

(A) Diagram depicting probing of NMDARs with a rapid-exchange system [28]: a neutrophil (held in whole-cell) is stimulated pharmacologically by applying different solutions through two channels of ϴ-glass pipette (tip diameter ∼200 μm) mounted on a piezo-drive to enable the ultra-fast delivery (<1 ms resolution; solutions in ϴ glass exchanged within 10 s using a rapid multi-channel perfusion system); see Videos S1 and S2 for live videos of neutrophils before and during patching.

(B) Representative whole-cell currents (patch pipette 4–7 MΩ, 1–2 μM tip) recorded in human neutrophils in response to locally applied NMDA (1 mM) and glycine (1 mM), in zero Mg²⁺, 2 mM Mg²⁺, and in the presence of NMDAR antagonists APV (50 μM) and Co101244 (1 μM) in zero Mg²⁺ (control), as indicated; same-cell pharmacological manipulations applied at ∼20 s intervals.

(C) Summary of experiments shown in (B); mean ± SEM (amplitude over the 300–500 ms pulse segment), normalized to control (sample size shown); ∗∗∗p < 0.01. Inset, super-resolution dSTORM image of a neutrophil shown with chromatically separated GluN2B and elastase single-molecule labels as indicated; see Figure S1A for further detail and illustrations.

(D) Characteristic images of a neutrophil (gray DIC image, top raw) loaded with CellTracker Red (red channel, middle) and Fluo 4-AM (green, bottom), in baseline conditions, after bath application of NMDA (100 μM, 2-3-min duration) and glycine (50 μM), and after adding PMA (1 μM) for Ca²⁺ homeostasis control, as indicated; experimental timing as shown; false color scale: relative intensity, arbitrary units (au); pixel size ∼120 nm (near diffraction limit).

(E) Experiment as in (D), but in the presence of the selective GluN2B-containing NMDAR antagonist Co101244 (1 μM); other notations as in (D).

(F) Statistical summary of experiments shown in (D and E). Average Ca²⁺ responses (mean ± SEM) of individual neutrophils, first to NMDA+Gly application (blue) and next to PMA (red) application, in either of the three solutions: 0 Mg²⁺ (as in D), 2 mM Mg²⁺, and 0 Mg²⁺ with Co101244 (as in E), as indicated; no NMDA+Gly was applied to control sample (Cntrl, green; 0 Mg²⁺), to evaluate an experiment-wise drift in Ca²⁺-dependent fluorescence at the time of NMDA+Gly response measurement in other groups (dotted line); numbers, sample size (in 0 Mg²⁺ group, 3 cells were lost at the PMA stage); ∗∗∗p < 0.001 (paired t test; two-sample t test for 0 Mg²⁺ group).

(G) One-cell example (neutrophil held in whole-cell, patch pipette is seen) showing that depolarization current induces prominent Ca²⁺ mobilization; inset, DIC+Fluo-4 AM image; traces: F, Fluo-4 fluorescence signal; I_h, holding current.

(H) Summary of experiments shown in (G). Left: Snapshots (Fluo-4 channel) of the stimulated cell (red dot; a neighboring neutrophil can be seen), in control conditions (Cntrl) and in the presence of APV, as indicated, at the time points as indicated (seconds) with respect to the stimulus onset. Note that in control conditions, but not under APV, the neighboring cell responds with a Ca²⁺ rise. Right: Summary of depolarization-induced [Ca²⁺]_in rises in the patched cell, represented by the Fluo-4 ΔF/F₀ signal; dots, individual cells; bars: mean ± SEM; sample size shown.