Hyperpolarization-activated (pacemaker) channels are dually gated by negative voltage and intracellular cAMP. Kinetics of native cardiac f-channels are not compatible with HH gating, and require closed/open multistate models. We verified that members of the HCN channel family (mHCN1, hHCN2, hHCN4) also have properties not complying with HH gating, such as sigmoidal activation and deactivation, activation deviating from fixed power of an exponential, removal of activation “delay” by preconditioning hyperpolarization. Previous work on native channels has indicated that the shifting action of cAMP on the open probability (Po) curve can be accounted for by an allosteric model, whereby cAMP binds more favorably to open than closed channels. We therefore asked whether not only cAMP-dependent, but also voltage-dependent gating of hyperpolarization-activated channels could be explained by an allosteric model. We hypothesized that HCN channels are tetramers and that each subunit comprises a voltage sensor moving between “reluctant” and “willing” states, whereas voltage sensors are independently gated by voltage, channel closed/open transitions occur allosterically. These hypotheses led to a multistate scheme comprising five open and five closed channel states. We estimated model rate constants by fitting first activation delay curves and single exponential time constant curves, and then individual activation/deactivation traces. By simply using different sets of rate constants, the model accounts for qualitative and quantitative aspects of voltage gating of all three HCN isoforms investigated, and allows an interpretation of the different kinetic properties of different isoforms. For example, faster kinetics of HCN1 relative to HCN2/HCN4 are attributable to higher HCN1 voltage sensors' rates and looser voltage-independent interactions between subunits in closed/open transitions. It also accounts for experimental evidence that reduction of sensors' positive charge leads to negative voltage shifts of Po curve, with little change of curve slope. HCN voltage gating thus involves two processes: voltage sensor gating and allosteric opening/closing.

The hyperpolarization-activated cyclic nucleotidegated (HCN) family of "pacemaker" channels includes 4 isoforms, the kinetics and cAMP-induced modulation of which differ quantitatively. Because HCN isoforms are highly homologous in the central region, but diverge more substantially in the N and C termini, we asked whether these latter regions could contribute to the determination of channel properties. To this aim, we analyzed activation/deactivation kinetics and the response to cAMP of heterologously expressed isoforms mHCN1 and rbHCN4 and verified that mHCN1 has much faster kinetics and lower cAMP sensitivity than rbHCN4. We then constructed rbHCN4 chimeras by replacing either the N or the C terminus, or both, with the analogous domains from mHCN1. We found that: 1) replacement of the N terminus (chimera N1-4) did not substantially modify either the kinetics or cAMP dependence of wildtype channels; 2) replacement of the C terminus, on the contrary, resulted in a chimeric channel (4-C1), the kinetics of which were strongly accelerated compared with rbHCN4, and that was fully insensitive to cAMP; 3) replacement of both N and C termini led to the same results as replacement of the C terminus alone. These results indicate that the C terminus of rbHCN4 contributes to the regulation of voltage-and cAMP-dependent channel gating, possibly through interaction with other intracellular regions not belonging to the N terminus.Together with voltage-dependent K ϩ channels and cyclic nucleotide-gated channels, the recently cloned hyperpolarization-activated cyclic nucleotide-gated (HCN) 1 channels belong to the superfamily of 6 transmembrane domain channels (1). HCN channel subunits are the molecular building blocks of native hyperpolarization-activated "pacemaker" (f/h) channels of cardiac and neuronal cells (2, 3). A peculiar property of HCN channels is their dual modulation by voltage hyperpolarization and by intracellular cAMP (4); as in voltage-dependent K ϩ channels, voltage sensing is localized in the S4 transmembrane domain (5, 6), and as in cyclic nucleotide-gated channels, gating by cAMP requires direct binding of cAMP molecules to the cyclic nucleotide binding (CNB) domain located intracellularly at the C terminus (7).Thus far, four HCN isoforms (1-4) have been cloned. When expressed heterologously, HCN channels display kinetic and modulatory properties similar to those of native I f /I h channels (8 -14). However, different isoforms have qualitatively similar, but quantitatively different properties. Activation and deactivation kinetics for example are much faster for HCN1 than HCN2 or HCN4, whereas the HCN1 sensitivity to cAMP is much reduced; further, the activation threshold of HCN2 is more negative than that of either HCN1 or HCN4 (15, 16).These differences are important in that they can provide a molecular basis for the diverse properties of native channels in various cell types; it is known for example that the I h current of hippocampal CA1 neurons has faster kinetics and a reduced cAMP sensitivity relativ...