Extended Data Fig. 5: Perfusion of propofol to HCN1 M305L recovers voltage dependent gating.
From: Propofol rescues voltage-dependent gating of HCN1 channel epilepsy mutants
a, Schematic of perfusion experiment design. Two electrode voltage clamp recordings were performed pre- and post-perfusion with 30 µM propofol for 10 min. To verify inward HCN1 currents, recording solution supplemented with 1 mM caesium chloride was perfused on and off the cell for 5 min. Shown are representative traces of n = 3 and 4 similar recordings with b, WT and c, M305L, respectively. d, For WT and M305L, the inward current is blocked by caesium while the outward depolarized tails remain intact. Corresponding Boltzmann fits are also shown for WT and M305L. Controls demonstrating inward current caesium block in the absence of 30 µM propofol are shown for e, WT and f, M305L and are representative traces of n = 3 similar recordings. Voltage clamp ranged from +45 mV to −125 mV with tail currents measured at +50 mV. The current response at −85 mV is highlighted in red. Empty and filled symbols with error bars represent mean ± s.d. for normalized apo and propofol data, respectively. n represents the number of biological replicates. HEK293S GnTI- cells transfected with HCN1 g, WT and h, M305L using Lipofectamine 2000 (Invitrogen). Nuclei are in blue, the plasma membrane in red, and HCN1 in green. Expression at the plasma membrane is demonstrated by colocalization (yellow). Shown is a representative cell of WT n = 20 and M305L n = 10 similar cells, over 3 independent transfections. Plotted to the right are intensity values across the dashed orange line. The scale bar represents 10 µm. For microscopy source data, see Supplementary Fig. 2.
