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Members of the hyperpolarization-activated cation (HCN) channel family generate HCN currents (I(h)) that are directly regulated by cAMP and contribute to pacemaking activity in heart and brain. The four different HCN isoforms show distinct biophysical properties. In cell-free patches from Xenopus oocytes, the steady-state activation curve of HCN2 channels is 20 mV more hyperpolarized compared with HCN1. Whereas the binding of cAMP to a COOH-terminal cyclic nucleotide binding domain (CNBD) markedly shifts the activation curve of HCN2 by 17 mV to more positive potentials, the response of HCN1 is much less pronounced (4 mV shift). A previous deletion mutant study suggested that the CNBD inhibits hyperpolarization-gating in the absence of cAMP; the binding of cAMP shifts gating to more positive voltages by relieving this inhibition. The differences in basal gating and cAMP responsiveness between HCN1 and HCN2 were proposed to result from a greater inhibitory effect of the CNBD in HCN2 compared with HCN1. Here, we use a series of chimeras between HCN1 and HCN2, in which we exchange the NH(2) terminus, the transmembrane domain, or distinct domains of the COOH terminus, to investigate further the molecular bases for the modulatory action of cAMP and for the differences in the functional properties of the two channels. Differences in cAMP regulation between HCN1 and HCN2 are localized to sequence differences within the COOH terminus of the two channels. Surprisingly, exchange of the CNBDs between HCN1 and HCN2 has little effect on basal gating and has only a modest one on cAMP modulation. Rather, differences in cAMP modulation depend on the interaction between the CNBD and the C-linker, a conserved 80-amino acid region that connects the last (S6) transmembrane segment to the CNBD. Differences in basal gating depend on both the core transmembrane domain and the COOH terminus. These data, taken in the context of the previous data on deletion mutants, suggest that the inhibitory effect of the CNBD on basal gating depends on its interactions with both the C-linker and core transmembrane domain of the channel. The extent to which cAMP binding is able to relieve this inhibition is dependent on the interaction between the C-linker and the CNBD.
Figure 1. Comparison of hyperpolarization-activated currents and response to cAMP for HCN1, HCN2, and their COOH-terminal chimeras. Icons in the top row represent wild-type HCN1 (sequences represented by bold lines, leftmost icon), HCN2 (sequences represented by light lines, rightmost icon), and their COOH-terminal chimeras HCN112 and HCN221. (A) Currents elicited by 3-s hyperpolarizations to indicated potentials from inside-out patches obtained from oocytes injected with cRNA of (from left to right) HCN1, HCN112, HCN221, or HCN2. Patches were stepped to voltages ranging from â85 to â165 mV in 10-mV steps from a holding potential of â40 mV. (B) Currents shown for same patches in the presence of 10 μM cAMP in internal (bath) solution. (C) Comparison of activation kinetics during a step to â135 mV in the presence and absence of 10 μM cAMP. Superimposed traces from records shown in A and B were scaled so that the current amplitudes appear equal to show the differences in activation kinetics.
Figure 2. Tail current activation curves for HCN1, HCN2, and COOH-terminal chimeras in the absence and presence of cAMP. Mean, normalized tail current amplitudes (materials and methods) are plotted on the y-axis as a function of test voltage. Solid curves show fitted Boltzmann relation. (closed symbols) In the presence of cAMP; (open symbols) in the absence of cAMP. Bars indicate SEM. (A) HCN1. V1/2 and s values from fitted Boltzmann relations are, respectively, â115.3 and 6.2 mV in the absence of cAMP and â111.2 and 5.0 mV in the presence of cAMP (n = 7 patches). (B) HCN2. V1/2 and s equal â135.7 and 5.1 mV, respectively, in the absence of cAMP, and â118.8 and 5.2 mV in the presence of cAMP, respectively (n = 10). (C) HCN221. V1/2 and s values from fitted Boltzmann relations are, respectively, â121.8 and 5.5 mV in the absence of cAMP and â116.2 and 5.1 mV in the presence of cAMP (n = 6). (D) HCN112. V1/2 and s values from fitted Boltzmann relations are, respectively, â124.4 and 4.8 mV in the absence of cAMP and â105.7 and 5.8 mV in the presence of cAMP (n = 10 patches).
Figure 4. Basal voltage dependence and cAMP modulation of wild-type HCN1, HCN2, and their NH2-terminal, transmembrane domain, and COOH-terminal chimeras. Icons at left represent HCN1 and HCN2 channels sequences as bold and light lines, respectively. Data plotted next to each channel show steady-state V1/2 in the absence of cAMP (left graph) and magnitude of shift in V1/2 in response to 10 μM cAMP (right graph); bars indicate SEM. Mean V1/2 values, slope values, and number of patches are in Table .
Figure 5. Contribution of COOH-terminal subdomains to differences in basal voltage dependence of activation between HCN1 and HCN2. (A). Effect of replacing different regions of the COOH terminus of HCN1â² with corresponding regions of HCN2. V1/2 of chimeras are plotted relative to the V1/2 of HCN1â² (i.e., V1/2 of a given channel minus V1/2 of HCN1â²). Note the negative voltage scale (indicating negative shift in V1/2 for a given chimera relative to HCN1â²). (B) Effect of replacing different regions of the COOH terminus of HCN2 with corresponding regions of HCN1. Note the positive voltage scale (indicating positive shift in V1/2 for a given chimera relative to HCN2). Mean values for V1/2, slope of Boltzmann fit, and number of patches for HCN1â² and the COOH-terminal chimeras are in Table . Data for HCN2, HCN221, and HCN112 are in Table .
Figure 6. The C-linker has no independent effect on voltage dependence of gating. (A) Schematic drawings of HCN1ÎC-term and HCN1â2LÎCNBD (bold line indicates HCN1 and light line HCN2). (B) Steady-state tail current activation curves for HCN1ÎC-term (solid curve, open circles) and HCN1â2LÎCNBD (dashed curve, closed squares). The curves show the fits of Boltzmann relations that yield the following parameters: for HCN1ÎC-term, V1/2 = â110.5 mV and s = 5.8 mV (11 patches); and for HCN1â2LÎCNBD, V1/2 = â109.1 mV and s = 7.4 mV (4 patches). (C) Difference between the V1/2 of HCN1ÎC-term, HCN1â2LÎCNBD, or HCN1ÎCNBD (data from Wainger et al. 2001) and the V1/2 of wild-type HCN1 (i.e., V1/2 of a given deletion mutant minus the V1/2 of HCN1). Also shown is the shift in V1/2 of HCN1 by saturating [cAMP] (data in Table ). Error bars show SEM.
Figure 7. Contribution of COOH-terminal subdomains to differences in cAMP modulation between HCN1 and HCN2. (A) Change in response to cAMP upon replacement of COOH-terminal regions of HCN1â² with corresponding region of HCN2 (terminology as in Fig. 5). Maximal shift in V1/2 in response to 10 μM cAMP for a given channel is plotted relative to the maximal shift for HCN1â² (i.e., shift in V1/2 in response to cAMP for a given chimera minus the shift for HCN1â²). Positive values indicate that a given construct shows a larger positive shift in response to cAMP compared with HCN1â². (B) Change in response to cAMP upon replacement of COOH-terminal regions of HCN2 with corresponding region of HCN1. Maximal shift in V1/2 in response to 10 μM cAMP for a given channel is plotted relative to the maximal shift for HCN2 (i.e., shift in V1/2 for a chimeric channel minus the shift for HCN2). Negative values indicate that a given construct shows a smaller positive shift in response to cAMP compared with HCN2. Mean values for the voltage shifts and the number of patches for HCN1â² and the COOH-terminal chimeras are in Table . Data for HCN2, HCN221, and HCN112 are in Table .
Figure 3. cAMP doseâresponse curves for HCN1, HCN2, and COOH-terminal chimeras. Shifts in V1/2 (ÎV1/2) are plotted as a function of [cAMP]. Solid lines show fits of Hill equation (materials and methods). Bars indicate SEM. (A) HCN1. Fit of the Hill equation yields: maximal shift = 4.1 mV, K1/2 = 0.06 μM, and h = 1.0 (n = 26 patches). (B) HCN2. Maximal shift = 17.4 mV, K1/2 = 0.10 μM, and h = 1.1 (n = 16). (C) HCN221. Maximal shift = 5.4 mV, K1/2 = 0.02 μM, and h = 1.4 (n = 14). (D) HCN112. Maximal shift = 20.4 mV, K1/2 = 0.07 μM, and h = 1.1 (n = 17).
Broillet,
Direct activation of the olfactory cyclic nucleotide-gated channel through modification of sulfhydryl groups by NO compounds.
1996, Pubmed
Broillet,
Direct activation of the olfactory cyclic nucleotide-gated channel through modification of sulfhydryl groups by NO compounds.
1996,
Pubmed
Chen,
Functional roles of charged residues in the putative voltage sensor of the HCN2 pacemaker channel.
2000,
Pubmed
,
Xenbase
Chen,
Properties of hyperpolarization-activated pacemaker current defined by coassembly of HCN1 and HCN2 subunits and basal modulation by cyclic nucleotide.
2001,
Pubmed
,
Xenbase
del Camino,
Blocker protection in the pore of a voltage-gated K+ channel and its structural implications.
2000,
Pubmed
DiFrancesco,
Pacemaker mechanisms in cardiac tissue.
1993,
Pubmed
Gordon,
A histidine residue associated with the gate of the cyclic nucleotide-activated channels in rod photoreceptors.
1995,
Pubmed
,
Xenbase
Gordon,
Direct interaction between amino- and carboxyl-terminal domains of cyclic nucleotide-gated channels.
1997,
Pubmed
Gordon,
Localization of regions affecting an allosteric transition in cyclic nucleotide-activated channels.
1995,
Pubmed
,
Xenbase
Goulding,
Molecular cloning and single-channel properties of the cyclic nucleotide-gated channel from catfish olfactory neurons.
1992,
Pubmed
Jan,
Cloned potassium channels from eukaryotes and prokaryotes.
1997,
Pubmed
Kaupp,
Molecular diversity of pacemaker ion channels.
2001,
Pubmed
Ludwig,
A family of hyperpolarization-activated mammalian cation channels.
1998,
Pubmed
Ludwig,
Two pacemaker channels from human heart with profoundly different activation kinetics.
1999,
Pubmed
Paoletti,
C-Linker of cyclic nucleotide-gated channels controls coupling of ligand binding to channel gating.
1999,
Pubmed
Pape,
Queer current and pacemaker: the hyperpolarization-activated cation current in neurons.
1996,
Pubmed
Perozo,
Structural rearrangements underlying K+-channel activation gating.
1999,
Pubmed
Santoro,
Molecular and functional heterogeneity of hyperpolarization-activated pacemaker channels in the mouse CNS.
2000,
Pubmed
,
Xenbase
Santoro,
Identification of a gene encoding a hyperpolarization-activated pacemaker channel of brain.
1998,
Pubmed
,
Xenbase
Santoro,
The HCN gene family: molecular basis of the hyperpolarization-activated pacemaker channels.
1999,
Pubmed
Santoro,
Interactive cloning with the SH3 domain of N-src identifies a new brain specific ion channel protein, with homology to eag and cyclic nucleotide-gated channels.
1997,
Pubmed
Seifert,
Molecular characterization of a slowly gating human hyperpolarization-activated channel predominantly expressed in thalamus, heart, and testis.
1999,
Pubmed
Shin,
Blocker state dependence and trapping in hyperpolarization-activated cation channels: evidence for an intracellular activation gate.
2001,
Pubmed
Vaca,
Mutations in the S4 domain of a pacemaker channel alter its voltage dependence.
2000,
Pubmed
Varnum,
Interdomain interactions underlying activation of cyclic nucleotide-gated channels.
1997,
Pubmed
,
Xenbase
Wainger,
Molecular mechanism of cAMP modulation of HCN pacemaker channels.
2001,
Pubmed
Zagotta,
Structure and function of cyclic nucleotide-gated channels.
1996,
Pubmed
