Cyclic AMP binds to the HCN channel C terminus and variably stabilizes its open state. Using isothermal titration calorimetry, we show that cAMP binds to one subunit of tetrameric HCN2 and HCN4 C termini with high affinity (ϳ0.12 M) and subsequently with low affinity (ϳ1 M) to the remaining three subunits. Changes induced by high affinity binding already exist in both a constrained HCN2 tetramer and the unconstrained HCN1 tetramer. Natural "preactivation" of HCN1 may explain both the smaller effect of cAMP on stabilizing its open state and the opening of unliganded HCN1, which occurs as though already disinhibited.Binding of cAMP to the cytoplasmic side of the HCN channel makes it easier to open (1); in the heart, this speeds rate and enhances conduction (2). In the sinoatrial node, opening is facilitated with a half-maximal cAMP concentration of 0.2 M (1) and is marked by a depolarizing shift in the voltage range over which the channel opens (3). Each of four mammalian HCN isoforms (HCN1-4) (4 -7) contains a cyclic nucleotidebinding domain (CNBD) 3 in the cytosolic C terminus and has significant sequence homology to voltage-gated potassium channel subunits. Thus, HCNs are predicted to have six transmembrane helices (S1-S6) with a cytosolic N and C terminus and to combine as tetramers to form the ion-conducting channel containing four equivalent cAMP-binding sites.The structure of a fragment of the HCN2 C terminus was solved by x-ray crystallography, which showed that this structure forms a symmetrical tetramer (8). The structure, hanging below the pore, is derived from sequences that begin just after the end of the S6 transmembrane segment and includes both the CNBD and the C-linker, a region that connects the CNBD to the inner side of the channel pore. Cyclic AMP is thought to bind and induce a conformational change that propagates through the CNBD as well as to the C-linker of an adjacent subunit (9, 10), by interactions of select ␣ helices, to form a "gating ring" that disinhibits channel opening (8,11,12). Evidence for the formation of a gating ring comes from studies of the purified HCN2 C terminus using analytical ultracentrifugation (8). In the absence of cAMP, monomers are predominantly seen, whereas the proportion of multimers, especially tetramers, increases significantly when cAMP is present.Curiously, facilitation of opening by cAMP differs among the mammalian HCNs. HCN2 and HCN4 opening is strongly facilitated by cAMP, whereas that of HCN1 and HCN3 is only weakly affected or not affected at all, respectively (12, 13). This variation among the isoforms is thought to arise, at least in part, from subtle differences in primary structure of the CNBD and the C-linker (12,14,15). Notably, opening is facilitated even in chimeric HCN2 and HCN4 channels containing the CNBDs from HCN1 and HCN3 (14,15), suggesting that all HCNs bind cAMP. Nevertheless, the molecular basis for the differences in cAMP effect among the isoforms remains unknown. Here, we set out to directly quantify and compare the energetics o...
EA. Regulation of cell surface expression of functional pacemaker channels by a motif in the B-helix of the cyclic nucleotide-binding domain. Am J Physiol Cell Physiol 295: C642-C652, 2008. First published July 9, 2008 doi:10.1152/ajpcell.00062.2008.-Previous studies have suggested that a portion of the cyclic nucleotide-binding domain (CNBD) of the hyperpolarization-activated cyclic nucleotide-gated channel 2 (HCN2) "pacemaker" channel, composed of the A-and B-helices and the interceding -barrel, confers two functions: inhibition of channel opening in response to hyperpolarization and promotion of cell surface expression. The sequence determinants required for each of these functions are unknown. In addition, the mechanism underlying plasma membrane targeting by this subdomain has been limitedly explored. Here we identify a four-amino acid motif (EEYP) in the B-helix that strongly promotes channel export from the endoplasmic reticulum (ER) and cell surface expression but does not contribute to the inhibition of channel opening. This motif augments a step in the trafficking pathway and/or the efficiency of correct folding and assembly.pacemaker channel function; protein export; trafficking; hyperpolarization-activated cyclic nucleotide-gated channel HYPERPOLARIZATION-ACTIVATED cyclic nucleotide-gated "pacemaker" channels (HCN1-4) contribute to the regulation of spontaneous activity and membrane potential in mammalian cardiac conduction tissue and brain (21). The number of HCN channels on the cell surface is critical to these functions, but the factors that determine their supply to this region of the cell have not been extensively studied. In general, export of plasma membrane-bound ion channels from the endoplasmic reticulum (ER) to the Golgi is limited by multiple quality control mechanisms (4,6,7,22). The export of properly folded and assembled channels from the ER is also regulated and may depend on anterograde signals (6,14,15,17,24).In HCN2 channels, the cyclic nucleotide-binding domain (CNBD), located in the COOH terminus, appears to be an important determinant of cell surface expression, in addition to its better known role as regulator of channel opening (31), based on two studies (1,20). First, complex glycosylation is abolished in HCN2 mutants lacking the CNBD, which supports its necessity for export of the channel from the ER (1). Second, we identified a subdomain of the CNBD that strongly promotes cell surface and functional expression; mutants lacking this subdomain do not generate current and are retained intracellularly (20). This same subdomain, which consists of the A and B helices and the interceding -barrel, exerts tonic inhibition of channel opening in response to hyperpolarization (5). Whether the complete subdomain is required for both functions, perhaps by conferring a shared conformational change, or includes distinct regions that contribute to each function is not known.The mechanism by which the CNBD subdomain promotes cell surface expression is poorly understood. To date, it has b...
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