Figure 5: Membrane potential state alters cell membrane YFP-DAT levels.

(a) Simultaneous whole-cell patch-clamp recording with the patch pipette visible in dissolved inorganic carbon (top) of YFP-DAT HEK cell and TIRF imaging of YFP-DAT (white) of the same cell (bottom). (b) TIRF image sequence with 5-s interframe interval of YFP-DAT HEK cell footprint with the membrane potential clamped at indicated holding potentials. Hyperpolarized potentials (−40 to −60 mV transition) increase cell-surface YFP-DAT signal, while depolarized potentials (−40 to +20 mV transition) reduces cell-surface YFP-DAT within 5 s and recovers within 5–10 s on hyperpolarization (+20 to −60 mV transition). (c,f,i) The mean normalized intensity of the surface-membrane YFP-DAT over time for all clamped cells (solid line) held at −40 mV (c), −60 mV (f) and +20 mV (i) for 5 min. Error bars are omitted for clarity, and the mean intensity for matched corresponding adjacent non-patched cells (colour-matched dashed line) and the mean of all non-patched adjacent cells (grey line) are included for comparison. (d,g,j) Normalized fluorescence intensity values for patched-clamped (solid line) and paired adjacent cells (dotted line) before (0 min) and 3 min after membrane potential change (n=4–5 cells per group). (e,h,k) Representative images of YFP-DAT HEK TIRFM footprints of patched cells before (0 min), 3 and 5 min after the membrane potential change and 1 min after returning the membrane potential to −40 mV. (l) Average normalized YFP intensities at 5 min after the potential change to hyperpolarized (−60 mV, blue) or depolarized (+20 mV, red) membrane potentials are significantly different relative to continuous clamping near the endogenous (−40 mV, black) membrane potential. #P<0.05, **P<0.001 using independent samples t-test. All values are mean±s.e.m. Scale bars, 10 μm.