Journal of Physiology The ‘funny’ current (I f ), or cardiac pacemaker current, is highly expressed in the pacemaker cells of the sino-atrial node (SAN) and in other cardiac myocytes able to pace spontaneously, such as atrio-ventricular cells and Purkinje fibres of the conduction tissue. In SAN cells, I f drives slow diastolic depolarization and is responsible for generation of spontaneous activity. f-channels carry an inward current at diastolic voltages and are activated by membrane hyperpolarization and by direct binding of intracellular cAMP; the latter mechanism mediates heart rate changes induced by sympathetic and parasympathetic transmitters, which act by modifying cAMP synthesis by stimulation and inhibition of adenylate cyclase, respectively (DiFrancesco, 1993; Accili et al. 2002). The neuronal equivalent of I f , the hyperpolarization-activated current (I h ), is expressed in a variety of neuronal tissues where it serves different functions, all linked to its ability to induce a depolarizing action upon appropriate stimulation (Pape, 1996). I h can therefore control the spontaneous firing rate or modify cell excitability, this latter a function that can be especially relevant to modulation of synaptic strength or sensory detection (Demontis et al. 1999; Beaumont & Zucker, 2000; Stevens et al. 2001; Mellor et al. 2002). The molecular constituents of f- and h-channels have been identified with the cloning of a family of hyperpolarization- activated, cyclic nucleotide-gated (HCN) channels. Four different isoforms (HCN1–4) have been cloned from a variety of tissues, including cardiac and neuronal tissues (Santoro et al. 1997, 1998; Gauß et al. 1998; Ludwig et al. 1998; Vaccari et al. 1999). When expressed in heterologous (Ishii et al. 1999; Ludwig et al. 1999; Seifert et al. 1999; Moroni et al. 2000; Moosmang et al. 2001; Viscomi et al. 2001) and in homologous (Qu et al. 2001) systems, these channels exibit properties typical of I f /I h , although quantitative differences exist in both the kinetics and the cAMP sensitivity (Santoro & Tibbs, 1999; Altomare et al. 2001; Kaupp & Seifert, 2001). Typically, the HCN1 isoform has much faster kinetics and a much poorer cAMP sensitivity than the HCN2 and HCN4 isoforms. Heteromeric HCN1–HCN4 channels: a comparison with native pacemaker channels from the rabbit sinoatrial node Claudia Altomare *, Benedetta Terragni *, Chiara Brioschi *, Raffaella Milanesi *, Cinzia Pagliuca *, Carlo Viscomi *, Anna Moroni *†, Mirko Baruscotti * and Dario DiFrancesco *† * Department of General Physiology and Biochemistry, Laboratory of Molecular Physiology and Neurobiology and † INFM-Milano U. Unit, via Celoria 26, 20133 Milano, Italy ‘Funny-’ (f-) channels of cardiac sino-atrial node (SAN) cells are key players in the process of pacemaker generation and mediate the modulatory action of autonomic transmitters on heart rate. The molecular components of f-channels are the hyperpolarization-activated, cyclic nucleotide- gated (HCN) channels. Of the four HCN isoforms known, two (HCN4 and HCN1) are expressed in the rabbit SAN at significant levels. However, the properties of f-channels of SAN cells do not conform to specific features of the two isoforms expressed locally. For example, activation kinetics and cAMP sensitivity of native pacemaker channels are intermediate between those reported for HCN1 and HCN4. Here we have explored the possibility that both HCN4 and HCN1 isoforms contribute to the native I f in SAN cells by co-assembling into heteromeric channels. To this end, we used heterologous expression in human embryonic kidney (HEK) 293 cells to investigate the kinetics and cAMP response of the current generated by co-transfected (HCN4 + HCN1) and concatenated (HCN4–HCN1 (4–1) tandem or HCN1–HCN4 (1–4) tandem) rabbit constructs and compared them with those of the native f-current from rabbit SAN. 4–1 tandem, but not co- transfected, currents had activation kinetics approaching those of I f ; however, the activation range of 4–1 tandem channels was more negative than that of the f-channel and their cAMP sensitivity were poorer (although that of 1–4 tandem channels was normal). Co-transfection of 4–1 tandem channels with minK-related protein 1(MiRP1) did not alter their properties. HCN1 and HCN4 may contribute to native f-channels, but a ‘context’-dependent mechanism is also likely to modulate the channel properties in native tissues. (Resubmitted 28 February 2003; accepted after revision 19 March 2003; first published online 17 April 2003) Corresponding author D. DiFrancesco: Department of General Physiology and Biochemistry, Laboratory of Molecular Physiology and Neurobiology and INFM- Milano U. Unit, via Celoria 26, 20133 Milano, Italy. Email: dario.difrancesco@unimi.it J Physiol (2003), 549.2, pp. 347–359 DOI: 10.1113/jphysiol.2002.027698 © The Physiological Society 2003 www.jphysiol.org