Figure 6.

Human pluripotent stem cells (hPSC)-sensory express a fast cAMP-insensitive I_h indicative of HCN1 ion channels. (a) Hyperpolarization-activated currents (I_h) were observed in hPSC-sensory when cells were voltage-clamped from a V_h of −60 mV to hyperpolarizing test voltage steps from −65 to −120 mV in 5 mV increments (bottom panel). Utilizing tail current analysis (see Materials and Methods), the V_1/2 of activation of I_h in hPSC-sensory was −84.7 ± 1.0 mV (n = 5). (b) Voltage relaxations (v-sag) typical of HCN-expressing neurons were observed in response to a hyperpolarizing injection in current-clamp mode (60 pA in this representative cell). (c) The average current density measured in voltage-clamp in response to a −120 mV voltage step was 31.8 ± 4.2 pA/pF (n = 10) in current-clamp mode an average of 0.3 ± 0.1 v-sag (n = 5) was observed in response to current injection achieving target voltages of ~−100 mV. (d) Activation kinetics were analyzed by fitting a double exponential to describe two activation rates (τ_fast and τ_slow). τ_fast kinetic analysis demonstrate a rapidly activating I_h similar to those observed in recombinant HCN1 ion channels. (e) Family of I_h traces in response to voltage steps as shown in a. In these examples, the cell was either measured in control conditions, forskolin-treated or in the presence of the I_h blocker cesium. (f) Voltage-activation curves for I_h were not affected following forskolin treatment (V_1/2 in control = −84.74 ± 1.0 mV and forskolin-treated = −84.0 ± 1.1 mV; n = 5; not significantly different). Likewise, current densities measured at −120 mV were not different between control (36.4 ± 6.8 pA/pF) and forskolin-treated (33.0 ± 5.6 pA/pF) cells. (g) I_h was completely blocked by a short application of the HCN channel blocker cesium (control = 31.8 ± 4.2 pA/pF and cesium = 0.9 ± 0.4 pA/pF, n = 10).