C Terminus-mediated Control of Voltage and cAMP Gating of Hyperpolarization-activated Cyclic Nucleotide-gated Channels

Abstract

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...

Interaction of the Pacemaker Channel HCN1 with Filamin A

“…Of the four HCN isoforms known, HCN1 is the least sensitive to cAMP, and shifts of the channel activation curve caused by saturating cAMP concentrations are of the order of only a few mV (4,11,32,41). Because we found that only HCN1 binds filamin A, this appears to contrast with the hypothesis that interaction with filamin A serves the purpose of concentrating elements of the cAMP second messenger cascade in restricted locations for improved channel cAMP-dependent modulation.…”

“…Heterologous expression of individual HCN isoforms does not normally result in currents whose properties reproduce entirely those of the native pacemaker (I f /I h ) currents (4,32). Part of this difference may be attributable to the existence of a "context" dependence of channel properties, i.e.…”

“…However, there is established evidence that the C terminus is involved in determining the kinetics of HCN channels (4,43,44), and the slowing due to the binding of the C terminus to filamin A might reflect an interference with the mechanism by which the C termini affect gating.…”

“…The molecular correlates of f/h channels are the hyperpolarizationactivated cyclic nucleotide-gated channels, of which four isoforms (HCN1-4) are known. When expressed alone in heterologous systems, HCN channels elicit currents whose properties are qualitatively similar to those of native I f /I h currents, although they differ quantitatively in kinetics and cAMP sensitivity (4,5).…”

Abstracts of Papers at the Fifty-Sixth Annual Meeting of the Society of General Physiologists

“…The structural analysis of HCN channels Shin et al, 2001;Viscomi et al, 2001;Wainger et al, 2001;Wang et al, 2001) points to the involvement of the COOH terminus and the S6 segments as physical domains involved in channel gating. Thus, trapping of the drug could be associated with physical occlusion of a large intracellular channel vestibule by the interaction between these and possibly other channel domains.…”