Cation conductance of HCN channels at extreme negative voltages. Membrane currents of HEK293 cells constitutively expressing HCN2 were measured in whole-cell configuration in buffers with different cations. Cells were clamped to a two-stage voltage protocol (top panel) comprising of 712- or 720-ms-long voltage steps from resting voltage to either −40 mV (black) or −130 mV (orange), respectively, followed by a fast ramp (12 mV/ms) from −40 or −130 to −250 mV. (A, C, and E) Membrane currents elicited in individual HCN2 expressing HEK293 cells by voltage protocol with prepulse to −40 mV (black) and −130 mV (orange) in standard buffer (110 mM NaCl/30 mM KCl; A), 140 mM CsCl (C), and 140 mM LiCl (E). Boxed currents in C and E are magnified in insets. (B, D, and F) Mean ∆I_HCN/V relationships in standard buffer (B), 140 mM CsCl (D), and 140 mM LiCl (F). ∆I/V data were obtained by subtracting ramp currents following prepulse to −40 mV from respective currents after prepulse to −130 mV after normalizing to currents at −130 mV. Black data points are means ± SD from HNC2 expressing HEK293 cells in standard medium (n = 10), 140 mM CsCl (n = 15), and 140 mM LiCl (n = 5). Blue data points are mean I/V relations (n = 7) obtained with same procedure as in C and D, but in control HEK293 cells not expressing HCN channels. Currents in D between −220 and −250 mV are significantly different (P < 0.001) between HCN2 expressing cells and non-expressing control cells.