Fig. 6: SR Kir2.1 channels are functional and show rectification.
a. Schematic representations (left) and phase contrast micrograph (right) of an isolated SR vesicle attached to the nucleus of a cardiomyocyte from normal non-infected mice in a patch-clamp experiment. This scheme was performed and considered representative after 30 perinuclear vesicles recorded from 14 animals. b, Representative patch-clamp recordings obtained by applying the voltage ramp protocol shown on top in the absence (black) and presence of 2 mM MgATP. Graphs show the magnitude currents in absolute values (left; P = 0.0267) and normalized to each control experiment (right, P = 0.0032). c, Representative patch-clamp recordings obtained by applying the voltage protocol shown on top in the absence (left) and presence (right) of spermine 10 µM. d, I–V relationships constructed at the end of the test pulses in b for control (black) and spermine (red). e, Time constant (τ) of block by spermine 10 µM (obtained from c) saturates at negative membrane potentials. Inset shows the same spermine data (red) compared to control (black). τ values were estimated using monoexponential fits. f–h, Left, representative patch-clamp experiment recordings using the voltage protocol shown on top. Right, time constant (τ) of block shown in left panels. f, Effect of residual endogenous spermine present in the SR vesicles under voltage-clamp conditions with symmetrical versus asymmetrical K+ concentration. g, Effect of spermine 10 µM after asymmetrical patch-clamping. h, Effect of caffeine 10 mM. τ values were estimated using monoexponential fits. i, Left, representative recordings from HEK293 cells transfected with Kir2.1 channels before and after perfusion with caffeine 10 mM. Right, quantification of caffeine-induced block at −120mV (P = 0.0175). Each value is represented as mean ± s.e.m. Statistical analyses were conducted using two-tailed t-test and two-way ANOVA test. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.
