The cardiac ‘funny’ (If) current of the sino-atrial node (SAN) is typical of pacing tissue and plays a key role in the generation and autonomic-mediated modulation of cardiac rate. Of the four isoforms of the recently cloned hyperpolarization-activated, cyclic-nucleotide-gated (HCN) channel family, three (HCN1, HCN2 and HCN4) have been found to be expressed in cardiac tissues. These HCN isoforms are heterogeneously distributed in the heart. HCN2 appears to be a predominant isoform in cardiac muscle; in the mammalian SAN, on the other hand, where f-channels are physiologically most relevant, RNase-protection assay analysis and immunolabelling show significant expression of HCN4 and HCN1, whereas mRNA levels of HCN2 are just detectable (Shi et al. 1999; Moroni et al. 2001).
The properties of HCN channels expressed in heterologous systems are similar to those of the native If current and of its neuronal equivalent Ih, but substantial quantitative differences exist among isoforms. Typically, HCN1 has faster activation/ deactivation kinetics, and a lower cAMP sensitivity than HCN2 and HCN4; HCN2 has in turn faster kinetics than HCN4; also, the voltage range of activation, and therefore the range where the pacemaker current is functionally important, varies among isoforms (Altomare et al. 2001). This is relevant to the question of the composition of native channels, since differences in the properties of native If /Ih currents in various tissues (DiFrancesco, 1993) may simply reflect an heterogeneous distribution of HCN isoforms.
In some cell types, however, the properties of native channels do not appear to conform to those of any of the individual isoforms locally expressed.
In the mammalian SAN, the reported activation kinetics of If are slower than those of heterologously expressed HCN1, and faster than those of HCN4. This leads to the idea that different HCN isoforms can co-assemble, to produce heteromers with properties intermediate between those of the individual components. Co-assembly of different HCN isoforms has indeed been demonstrated previously for HCN1 and HCN2 (Chen et al. 2001). HCN channels could also be modulated by auxiliary proteins. In the SAN, for example, co-transfection with the MiRP1 β-subunit has been reported to enhance expression and accelerate activation of HCN1 and HCN2 in Xenopus oocytes (Yu et al. 2001).
We used heterologous expression to understand if co-assembly of the major isoform components of HCN channels in SAN cells, HCN1 and HCN4, generates channels with the same properties as native SAN If channels. We investigated the kinetics and cAMP response of the current generated by cotransfected (rbHCN4 + rbHCN1) and concatenated (rbHCN4-rbHCN1, ‘tandem’) rabbit constructs expressed in HEK293 cells, and compared them with those of the native cardiac pacemaker current from the rabbit SAN.
We found that transfection in HEK293 cells of concatenated ‘tandem’ HCN1 and HCN4 isoforms generates pacemaker channels with activation kinetics approaching those of native f-channels in the SAN. However, we found no evidence for MiRP1 mediated modulation of expression and/or kinetics. Individual and cotransfected isoforms all had activation ranges more negative than the range of native f-channels, and tandem channels responded poorly to cAMP. The results are compatible with the idea that native f-channels are heteromers of HCN1 and HCN4 subunits, but only under the assumption that a ‘context’-dependent mechanisms contributes to determination of the channel properties in native tissue.