Chen S et al. (2007), Voltage sensor movement and cAMP binding allost...

XB-ART-35602

J Gen Physiol 2007 Feb 01;1292:175-88. doi: 10.1085/jgp.200609585.

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Voltage sensor movement and cAMP binding allosterically regulate an inherently voltage-independent closed-open transition in HCN channels.

Chen S , Wang J , Zhou L , George MS , Siegelbaum SA .

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The hyperpolarization-activated cyclic nucleotide-modulated cation (HCN) channels are regulated by both membrane voltage and the binding of cyclic nucleotides to a cytoplasmic, C-terminal cyclic nucleotide-binding domain (CNBD). Here we have addressed the mechanism of this dual regulation for HCN2 channels, which activate with slow kinetics that are strongly accelerated by cAMP, and HCN1 channels, which activate with rapid kinetics that are weakly enhanced by cAMP. Surprisingly, we find that the rate of opening of HCN2 approaches a maximal value with extreme hyperpolarization, indicating the presence of a voltage-independent kinetic step in the opening process that becomes rate limiting at very negative potentials. cAMP binding enhances the rate of this voltage-independent opening step. In contrast, the rate of opening of HCN1 is much greater than that of HCN2 and does not saturate with increasing hyperpolarization over the voltage range examined. Domain-swapping chimeras between HCN1 and HCN2 reveal that the S4-S6 transmembrane region largely determines the limiting rate in opening kinetics at negative voltages. Measurements of HCN2 tail current kinetics also reveal a voltage-independent closing step that becomes rate limiting at positive voltages; the rate of this closing step is decreased by cAMP. These results are consistent with a cyclic allosteric model in which a closed-open transition that is inherently voltage independent is subject to dual allosteric regulation by voltage sensor movement and cAMP binding. This mechanism accounts for several properties of HCN channel gating and has potentially important physiological implications.

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Species referenced: Xenopus laevis
Genes referenced: camp hcn1 hcn2

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	Figure 1. Four-state and eight-state allosteric models for regulation of HCN2 opening by voltage and cAMP. (A) The four-state cyclic allosteric scheme for channel opening in the absence of cAMP. The vertical transitions represent the voltage-independent opening and closing reactions. The horizontal transitions are voltage-dependent activation steps that reflect voltage sensor movements. Î± and Î² are the voltage-dependent rate constants for activation and deactivation, respectively, for the closed state of the channel. Î±â² and Î²â² are the voltage-dependent rate constants for activation and deactivation, respectively, for the open state of the channel. g and h are the rate constants for opening and closing, respectively, for the activated state of the channel. gâ² and hâ² are the rate constants for opening and closing, respectively, for the resting state of the channel. (B) The eight-state cubic scheme for the regulatory effects of both cAMP and voltage on channel opening. The four unliganded states correspond to the vertices on the front face of the cube (in black), and undergo transitions identical to those shown in A. The four cAMP-bound states form the four vertices on the back face of the cube (in blue). Rate constants marked with asterisks are for the cAMP-bound state transitions. In general, these rate constants differ from those in the absence of ligand. Ligand binding (+cAMP) and unbinding steps are shown in red. Transitions and states lying on hidden edges and corners of the cube are drawn with dashed lines and lighter shades. The rate constants for the binding and unbinding reactions are not shown since these transitions do not occur under the conditions of our experiments, performed in either the absence of cAMP or a saturating concentration of ligand.
	Figure 2. Kinetics of HCN2 and HCN1 opening over a wide range of hyperpolarized voltages. (A1 and A2) Currents from inside-out patches obtained from oocytes injected with cRNA of HCN2 (A1) or HCN1 (A2), respectively. Currents were elicited by hyperpolarizing steps 4 s in length. Patches were stepped from a holding potential of â40 mV in 10-mV hyperpolarizing increments to test potentials ranging from â90 to â190 mV for HCN2 and from â70 to â170 mV for HCN1. Icons besides the HCN symbols represent the domain structures of HCN channels (intracellular N and C termini and the S1âS6 transmembrane domain) using solid rectangles for HCN2 and open rectangles for HCN1. (B) Relation between the rate of opening (obtained from reciprocal of the time constant of opening, Ï) for HCN2 (squares) and HCN1 (circles) versus hyperpolarizing test potential. Values of 1/Ï (units of 1/seconds) were measured by fitting the opening time course of HCN currents with a single exponential function following an initial delay. n = 8 for HCN1; n = 6 for HCN2. Standard error bars are shown when larger than the symbol. Note that we examined a slightly more restricted range of potentials for HCN1 than HCN2 due to the greater degree of patch instability at negative voltages from oocytes expressing HCN1.
	Figure 3. Role of HCN channel domains in determining the differences in opening kinetics between HCN1 and HCN2. Chimeras between wild-type HCN1 and HCN2 were made by swapping the N termini, transmembrane domains, or C termini between the two channels. Icons in each panel show approximate composition of different chimeras. Filled objects represent HCN2 sequences and open objects represent HCN1 sequences. Each graph plots mean binned values of 1/Ï as a function of normalized binned test voltage (see Materials and methods) for HCN2 (squares; n = 9), HCN1 (circles; n = 7), and given chimera (open triangles). Standard error bars are shown when larger than the symbol for both the 1/Ï and voltage axes (see Materials and methods). Note that HCN1 and HCN2 data in this and subsequent figures was obtained from a different series of experiments than shown in Fig. 2. (A) Chimeras in which the N terminus of HCN2 was replaced by the N terminus of HCN1 (122, left; n = 6) or in which the N terminus of HCN1 was replaced with the N terminus of HCN2 (211, right; n = 7). (B) Chimeras in which the C terminus of HCN2 was replaced by the C terminus of HCN1 (221, left; n = 8) or in which the C terminus of HCN1 was replaced with the C terminus of HCN2 (112, right; n = 8). (C) Chimeras in which the S1âS6 region of HCN2 was replaced by the corresponding region of HCN1 (212, left; n = 7) or in which the S1âS6 region of HCN1 was replaced with that of HCN2 (121, right; n = 8).
	Figure 4. Effect on opening kinetics of replacing the S1âS3 or S4âS6 transmembrane subdomains of HCN2 with corresponding regions of HCN1. (A) Currents elicited by hyperpolarizing steps for chimera in which the S1âS3 region of HCN2 was replaced with the corresponding region of HCN1 (chimera 21S1-32). (B) Currents elicited by hyperpolarizing steps for chimera in which the S4âS6 region of HCN2 was replaced with the corresponding region of HCN1 (chimera 21S4-62). (C) Relation between the rate of opening (1/Ï) and test potential for the chimera 21S1-32 (inverted open triangles; n = 7), chimera 212 (solid triangles, from Fig. 3 C), wild-type HCN1 (solid circles, from Fig. 3) and HCN2 (solid squares, from Fig. 3). (D) Relation between the rate of opening (1/Ï) and test potential for the chimera 21S4-62 (inverted open triangles; n = 7) and other constructs as described above.
	Figure 5. Effect on opening kinetics of replacing the S1âS3 or S4âS6 transmembrane subdomains of HCN1 with corresponding regions of HCN2. (A) Currents elicited by hyperpolarizing steps for chimera in which the S1âS3 region of HCN1 was replaced with the corresponding region of HCN2 (chimera 12S1-31). (B) Currents elicited by hyperpolarizing steps for chimera in which the S4âS6 region of HCN1 was replaced with the corresponding region of HCN2 (chimera 12S4-61). (C) Relation between the rate of opening (1/Ï) and test potential for the chimera 12S1-31 (inverted open triangles; n = 5). 1/Ï values of HCN1 (filled circles), HCN2 (filled squares), and 121 (filled triangles) from Fig. 3 are also plotted for comparison. (D) Relation between the rate of opening (1/Ï) and test potential for the chimera 12S4-61 (inverted open triangles; n = 13) and other constructs described above.
	Figure 6. The effect of cAMP and deletion of the CNBD on the opening kinetics of HCN2. Rates of opening (1/Ï) versus test potential for HCN2 are plotted in the absence (squares) or presence (open triangles; n = 9) of a saturating concentration of cAMP (10 Î¼M). Data are also shown for the HCN2ÎCNBD deletion mutant, in which the CNBD and all sequence C-terminal to the CNBD have been deleted (open squares; n = 16). Data for HCN1 (filled circles) are shown for comparison.
	Figure 7. Effect of voltage and cAMP on HCN2 tail current kinetics. (A) Illustration of voltage pulse protocol. Membrane was held at âˆ’40 mV, stepped to âˆ’140 mV for 3 s, and then stepped to a series of more positive potentials to measure tail currents. Left, entire time course of protocol. Right, protocol on an expanded time scale during tail current measurements. Time scales are shown in B. (B) HCN2 currents in absence of cAMP, at either a slow time scale showing the entire time course during opening and closing protocols (left) or an expanded time scale illustrating tail currents (right). (C) Currents obtained in presence of 10 Î¼M cAMP at a slow (left) and expanded (right) time scale. (D) Plot of 1/Ï„ for tail current decay (obtained from single exponential fits) as a function of voltage during tail current measurements, either in absence (open circles) or presence (filled circles) of cAMP. Bars show SE (n = 4).
	Figure 8. Fits of three-state and four-state gating schemes to HCN2 opening and closing kinetics. (A) Fits of the three-state scheme to normalized HCN2 currents in absence of cAMP. Left, HCN2 currents were measured in response to hyperpolarizing steps (10 s) from a holding potential of â40 mV to a series of test voltages in 10-mV increments (selected voltages indicated next to traces). Currents were converted to normalized open probabilities, f(t), shown as solid black traces (see Materials and methods). Dashed red traces show best fit of three-state model to data. Only the first 8 s of each trace are shown. Right, HCN2 tail currents obtained at different depolarized potentials (shown next to traces) following steps to â140 mV to open the channels. Tail currents were converted to normalized open probabilities (solid black traces). Predictions of three-state model shown as dashed red traces. Best-fit parameters with their lower and upper limits (see Materials and methods) were as follows: Î±0 = 3.2 Ã 10â6, range from 1.2 Ã 10â7 to 8.0 Ã 10â5 msâ1; sÎ± = 9.1, range from 8.0 to 11.6 mV; Î²0 = 415.8, range from 18 to 7,500 msâ1, sÎ² = 49.0, range is greater than 10.8 mV; g = 0.0024, range from 0.0016 to 0.004 msâ1, h = 3.8 Ã 10â4, range from 2.7 to 5.7 Ã 10â4 msâ1. See text for definition of parameters and Materials and methods for normalization procedure. The mean summed square error during fits is 0.0032 for current opening data and 5.0 for tail current data. (B) Fits of the four-state scheme to normalized HCN2 currents in absence of cAMP. Data and traces as in A. Best-fit parameters for the scheme are given in Table I. Note that the model provides a reasonable fit to both opening (left) and closing (right) kinetics. (C) Fits of four-state scheme to normalized HCN2 currents obtained in presence of 10 Î¼M cAMP. The model again provides a reasonable description of both opening and closing kinetics. Because of the cyclic nature of the scheme, the rate constant Î±â² (V) in fits of four-state schemes in B and C was calculated from the other rate constants to conform to microscopic reversibility.

References [+] :

Altomare, Integrated allosteric model of voltage gating of HCN channels. 2001, Pubmed

Altomare, Integrated allosteric model of voltage gating of HCN channels. 2001, Pubmed
Baruscotti, Physiology and pharmacology of the cardiac pacemaker ("funny") current. 2005, Pubmed
Bell, Changes in local S4 environment provide a voltage-sensing mechanism for mammalian hyperpolarization-activated HCN channels. 2004, Pubmed , Xenbase
Biel, Cardiac HCN channels: structure, function, and modulation. 2002, Pubmed
Chen, Properties of hyperpolarization-activated pacemaker current defined by coassembly of HCN1 and HCN2 subunits and basal modulation by cyclic nucleotide. 2001, Pubmed , Xenbase
Chen, Functional roles of charged residues in the putative voltage sensor of the HCN2 pacemaker channel. 2000, Pubmed , Xenbase
Chen, HCN subunit-specific and cAMP-modulated effects of anesthetics on neuronal pacemaker currents. 2005, Pubmed
Craven, Salt bridges and gating in the COOH-terminal region of HCN2 and CNGA1 channels. 2004, Pubmed , Xenbase
Decher, Voltage-dependent gating of hyperpolarization-activated, cyclic nucleotide-gated pacemaker channels: molecular coupling between the S4-S5 and C-linkers. 2004, Pubmed , Xenbase
DiFrancesco, Direct activation of cardiac pacemaker channels by intracellular cyclic AMP. 1991, Pubmed
DiFrancesco, Dual allosteric modulation of pacemaker (f) channels by cAMP and voltage in rabbit SA node. 1999, Pubmed
Goulding, Molecular cloning and single-channel properties of the cyclic nucleotide-gated channel from catfish olfactory neurons. 1992, Pubmed
HODGKIN, A quantitative description of membrane current and its application to conduction and excitation in nerve. 1952, Pubmed
Horrigan, Coupling between voltage sensor activation, Ca2+ binding and channel opening in large conductance (BK) potassium channels. 2002, Pubmed , Xenbase
Ishii, Determinants of activation kinetics in mammalian hyperpolarization-activated cation channels. 2001, Pubmed
Long, Voltage sensor of Kv1.2: structural basis of electromechanical coupling. 2005, Pubmed
Ludwig, Absence epilepsy and sinus dysrhythmia in mice lacking the pacemaker channel HCN2. 2003, Pubmed
Ludwig, A family of hyperpolarization-activated mammalian cation channels. 1998, Pubmed
Lüthi, H-current: properties of a neuronal and network pacemaker. 1998, Pubmed
Männikkö, Hysteresis in the voltage dependence of HCN channels: conversion between two modes affects pacemaker properties. 2005, Pubmed , Xenbase
Männikkö, Voltage-sensing mechanism is conserved among ion channels gated by opposite voltages. 2002, Pubmed , Xenbase
Pian, Regulation of gating and rundown of HCN hyperpolarization-activated channels by exogenous and endogenous PIP2. 2006, Pubmed , Xenbase
Robinson, Hyperpolarization-activated cation currents: from molecules to physiological function. 2003, Pubmed
Rothberg, Voltage-controlled gating at the intracellular entrance to a hyperpolarization-activated cation channel. 2002, 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
Shi, Distribution and prevalence of hyperpolarization-activated cation channel (HCN) mRNA expression in cardiac tissues. 1999, Pubmed
Shin, Inactivation in HCN channels results from reclosure of the activation gate: desensitization to voltage. 2004, Pubmed
Stieber, Molecular basis for the different activation kinetics of the pacemaker channels HCN2 and HCN4. 2003, Pubmed
Vaca, Mutations in the S4 domain of a pacemaker channel alter its voltage dependence. 2000, Pubmed
Vemana, S4 movement in a mammalian HCN channel. 2004, Pubmed , Xenbase
Viscomi, C terminus-mediated control of voltage and cAMP gating of hyperpolarization-activated cyclic nucleotide-gated channels. 2001, Pubmed
Wainger, Molecular mechanism of cAMP modulation of HCN pacemaker channels. 2001, Pubmed
Wang, Activity-dependent regulation of HCN pacemaker channels by cyclic AMP: signaling through dynamic allosteric coupling. 2002, Pubmed , Xenbase
Wang, Regulation of hyperpolarization-activated HCN channel gating and cAMP modulation due to interactions of COOH terminus and core transmembrane regions. 2001, Pubmed , Xenbase
Zagotta, Structural basis for modulation and agonist specificity of HCN pacemaker channels. 2003, Pubmed