Figure 6
Simulation of I h properties and co-expression of HCN1 and HCN4 subunits in HEK293 cells. (A) The simulated combination of a fast- and a slow-activating HCN subunit replicates the characteristic kinetic properties (fast activation and slow deactivation) at P0. Simulation of Ih conducted by a fast- and slow-activating HCN subunit (black trace) elicited by a −130 mV step for 2 seconds (bottom) superimposed onto a trace obtained by the same voltage protocol in a neocortical neuron at P0 (grey trace). Inset: the kinetic behavior is explained by the time-dependent contribution of a fast (blue line) and a slow (black line) component. (B) Families of HCN1- and HCN4-mediated Ih recorded from HEK293 cells co-expressing these subunits. Currents in the absence (top) and presence (bottom) of cAMP were activated from a holding potential of −40 mV to command potentials between −40 and −130 mV of 2 seconds length (inset). Tail currents were recorded after stepping back to −40 mV and showed a slow deactivation and tail current amplitudes were measured at the time point indicated by the dotted red line. (C) Voltage sensitivity of HCN1 and HCN4 subunits co-expressed in HEK293 cells. Activation curves in the presence (closed box) and absence (open box) of cAMP are depicted as mean ± SEM. For illustration purposes the population means were fitted using a Boltzmann function. For evaluation of the voltage sensitivity individual neurons were considered (see D). Intracellular cAMP clearly shifted the half-maximal activation voltage by approximately 16 mV. (D) Population data on half-maximal activation voltage V1/2 for the HCN1 and HCN4 mediated Ih showed the marked difference (P <0.01) between experimental conditions without (top) and with (bottom) 100 μM intracellular cAMP. (E) Representative current deactivation of HCN1 and HCN4 co-expressing HEK cells (black) without (top) and with 100 μM cAMP (bottom) displayed with an overlay of a single exponential fit in red. (F) Population data on Ih deactivation kinetics in HCN1- and HCN4-expressing HEK293 cells without (left) and with (right) 100 μM intracellular cAMP. (G) Subunit ratio determines Ih kinetics. Simulation of different ratios of fast and slow HCN subunits with two independent first-order kinetic models. Displayed fractions of the fast subunit were (inside to outside - arrow direction) 0, 0.30, 0.60, 0.90 and 1.00, respectively. Bottom: The simulation (black trace) was experimentally supported by a co-expression of HCN1 and HCN4 (grey trace) in HEK293 cells as in (B) that showed similar results.