Functional characterization of coexpressed HCN4 and HCN4-573X subunits in COS-7 cells. (a) Schematic representation of the expression construct used for coexpression experiments (not drawn to scale). Wild-type and mutant HCN4 cDNAs are located on the same plasmid (indicated with dotted lines); initiation codons of open reading frames are indicated with horizontal arrows. Structural elements such as promoters (P_CMV, P_EF-1α), IRES, and polyadenylation signals are shown in open boxes. The PCR products amplified from reverse-transcribed total RNA are indicated by a horizontal line above the HCN4 open reading frames. The location of the EciI restriction site is indicated by a vertical arrow. pA, polyadenylation signal. (b) Agarose gel electrophoresis of EciI-digested RT-PCR products amplified from total RNA of transiently transfected COS-7 cells. The wild-type fragment has a size of 353 bp and contains no EciI restriction site. In contrast, the 352-bp-long mutant PCR fragment (573X) is digested by EciI (see Figure 2c). The resulting two fragments (160 bp and 192 bp) are not separated under the chosen conditions. Shown are representative current traces of coexpressed wild-type and HCN4-573X subunits elicited by hyperpolarizing voltage steps from –40 mV to –120 mV in the absence (c) or presence (d) of 8-Br-cAMP. Voltage dependences of heteromeric wild-type/hHCN4-573X (e) conductances in the absence (black circles) or presence (white circles) of 1 mM 8-Br-cAMP are shown. The dashed lines indicate the respective Boltzmann fits of HCN4 (WT) with or without application of 8-Br-cAMP taken from Figure 4. Absence of error bars indicates errors smaller than the symbol size.