PKA shifts the voltage dependence of HCN4 in CHO cells. (A) Average voltage dependence of activation for HCN4 currents. Normalized tail current amplitudes are plotted as a function of prepulse voltage for protocols as shown in the inset with control intracellular solution (filled black circles), intracellular with 20 U/ml PKA (open red circles), or intracellular with PKA plus 10 µM PKI (open blue triangles). Midpoint activation voltages were −89.8 ± 1.7 mV in control, n = 7; −83.8 ± 1.5 mV in PKA, n = 9; and −90.0 ± 2.0 mV in PKA + PKI, n = 8. (Inset) Representative whole cell currents recorded from a CHO cell line stably expressing HCN4. Currents were elicited by hyperpolarizing voltage steps from −40 to −150 mV (in 10-mV increments), which ranged in length from 32.5 to 4.2 s (starting with the longest step at −40 mV and decremented by 2.575 s each step). This voltage protocol was used in an attempt to measure the steady-state voltage dependence of activation; however, note that activation was still incomplete at the more depolarized potentials. (B) Activation midpoints for HCN4 currents from Boltzmann fits to data in A. Asterisk indicates statistical significance (P < 0.05). NS, not significant (P > 0.05).