Electrophysiological properties of leucine zipper alanine mutants.A, representative current traces for wild-type HCN2 and alanine mutants recorded during 2-s pulses from a holding potential of −30 mV to potentials ranging from −30 to −140 mV in 10-mV decrements. Tail currents were measured at −130 mV. The dotted line represents zero current. The arrow indicates the instantaneous current component. B, relative currents measured at the end of a 2-s activating pulse to −130 mV, recorded 2 days after injection. Currents were normalized to wild-type HCN2 amplitude of every recording day. Wild-type HCN2 channel currents were 7.2 ± 0.4 μA (n = 46). The mean currents of L343A, L350A, L357A, and L364A were 2.5 ± 0.1 μA (n = 42), 1.3 ± 0.1 μA (n = 42), 0.8 ± 0.1 μA (n = 39), and 0.6 ± 0.1 μA (n = 36), respectively. C, Western blot of HCN2 channel proteins expressed in Xenopus oocytes after injection of the same amount of wild-type or mutant HCN2 cRNAs. HCN channels with an extracellular HA epitope were detected by an HA antibody and a peroxidase-linked secondary antibody. D, relative surface expression determined by single cell luminometry. Surface expression was normalized to wild-type surface expression (1.00 ± 0.08). E, activation curves for wild-type HCN2 and alanine mutants of the leucine zipper. Relative currents were fitted to a Boltzmann equation. The V½, slope factor, and min-Po values are given in Table 1. For L364A, currents were normalized to wild-type HCN2 currents at −130 mV. Error bars, S.E.; ***, p < 0.001; n.s., non-significant changes.