Fig. 1: Ca²⁺-clearance by PMCA2-NPTN in the millisecond-range.

a Surface-illustration (space-filling mode) of the PMCA2-NPTN complex with bound PIP₂ as determined in cryo-EM analyses¹⁶. b Steady-state activation curves of BK_Ca channels recorded in whole-cell mode with 10 µM free Ca²⁺ in the patch-pipette (insets) from CHO(ΔPMCA2)-cells in the absence (squares and line in black, 8 cells) and presence of heterologously expressed PMCA2-NPTN complexes (squares and line in red, 11 cells). Activation curves recorded with 1 µM and 0.1 µM free Ca²⁺ in the patch-pipette were added for calibration. Lines are result of a Boltzmann function fitted to the data (mean ± SEM). Note the large pump-mediated shift of the activation curve indicating a reduction of [Ca²⁺]_i from pipette-delivered 10 µM (dark grey) to values below 0.1 µM (light grey). c Representative BK_Ca-mediated outward K⁺ currents recorded with the indicated voltage-protocol in cultured CHO cells in response to a 0.8 ms Ca²⁺ influx through voltage-gated Cav2.2 channels in the presence of either 10 mM EGTA (trace in blue) or 0.1 mM EGTA in the absence (control, black trace) or presence of PMCA2-NPTN complexes (trace in red). Note the rapid current decay with 10 mM EGTA and with PMCA2-NPTN at 0.1 mM EGTA as a cytoplasmic buffer. d Current traces (in the framed box in (c)) at an expanded time scale, the decay phase approximated with a mono-exponential function with indicated time constant (τ). Currents were scaled to the maximum prior to the Ca²⁺ influx pulse. e Representative fluorescence traces recorded in 10-12 experiments with identical conditions as in (c), but with the membrane-tethered fluorescent Ca²⁺-indicator Lck-GCaMP6s used for monitoring changes in [Ca²⁺]_i (left insets). Gray line is the result of a mono-exponential fitted to the decay phase of the traces with PMCA2-NPTN and 10 EGTA. Right inset: Fluorescent signals (at the framed box) at an expanded time scale. f Representative BK_Ca-currents recorded in response to 1, 2 and 5 Ca²⁺ influx pulses applied at 500 Hz (voltage protocol in inset) to CHO cells as in (a). g Plot summarizing the time constants of the current decay determined in experiments as in (f); squares represent mean ± SEM of the indicated number of cells.