Fig. 2.
CDR and hysteresis of HCN4 control the firing mode of SAN cells. (A) Action potential recording of an isolated pacemaker cell showing the typical alternation between firing and nonfiring. The mean membrane potential is more depolarized during firing (~ − 55 mV, green line) and more hyperpolarized during nonfiring (~ − 75 mV, red line). At the same time, slow drifts in membrane potential occur. The firing mode is characterized by a slow, progressive hyperpolarization (Δ = − 7 mV) until firing stops (1), leading to an abrupt drop to significantly more hyperpolarized potentials. Conversely, during nonfiring, a slow and progressive depolarization occurs until the threshold for firing is reached (2), and the membrane potential abruptly jumps to substantially more depolarized values. Due to hysteresis of HCN4, the changes in membrane potential have important consequences for the voltage-dependent activation of the channel. (B) Original steady-state activation curves recorded from HCN4 channels heterologously expressed in HEK239 cells without cAMP in the intracellular solution. The long-lasting, mean membrane potentials during firing (− 55 mV) and nonfiring (− 75 mV) are mimicked by the holding potential (HP). At a relatively depolarized holding potential of − 55 mV, the activation curve is positioned at extremely hyperpolarized voltages (left curve). Conversely, at a relatively hyperpolarized holding potential of − 75 mV, the activation curve is positioned at extremely depolarized voltages (right curve). Points (1) and (2) on the activation curves reflect the time points (1) and (2) of the action potential measurements shown in panel (A). At the end of firing (1), the activation curve is shifted to the left and the membrane potential is relatively positive, resulting in a small number of open HCN4 channels. The consequent lack of a sufficiently depolarizing If current causes or supports the transition of pacemaker cells to the nonfiring mode. At the end of nonfiring (2), the activation curve is shifted to the right and the membrane potential is relatively negative, leading to a substantial increase in the number of open HCN4 channels, thereby causing or supporting the return of pacemaker cells to the firing mode. (C) Original activation curves of HCN4 channels recorded in the presence of 100 µM cAMP in the intracellular solution. Compared to panel (B), both curves are markedly shifted to the right. Under comparable conditions, SAN cells do not switch into the nonfiring mode, and the mean membrane potential permanently remains at depolarized values (− 55 mV), leading to a sufficient number of open HCN4 channels to maintain continuous firing