Published online before print June 6, 2002, doi: 10.1161/01.RES.0000024390.97889.C6
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© 2002 American Heart Association, Inc.
Molecular Medicine |
Dominant-Negative Suppression of HCN1- and HCN2-Encoded Pacemaker Currents by an Engineered HCN1 Construct
Insights Into Structure-Function Relationships and Multimerization
From the Institute of Molecular Cardiobiology, The Johns Hopkins University School of Medicine, Baltimore, Md.
Correspondence to Ronald A. Li, PhD, Assistant Professor of Medicine, Institute of Molecular Cardiobiology, The Johns Hopkins University School of Medicine, 720 Rutland Ave/Ross 844, Baltimore MD 21205. E-mail ronaldli{at}jhmi.edu
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Abstract |
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If, a diastolic depolarizing current activated by hyperpolarization, is a key player in cardiac pacing. Despite the fact that If has been known for over 20 years, the encoding genes, namely HCN1 to 4, have only recently been identified. Functional data imply that different HCN isoforms may coassemble to form heteromeric channel complexes, but little direct evidence is available. Subunit stoichiometry is also unknown. Although the pore region of HCN channels contains the glycine-tyrosine-glycine (GYG) signature motif found in K+-selective channels, they permeate both Na+ and K+. In the present study, we probed the functional importance of the GYG selectivity motif in pacemaker channels by replacing this triplet in HCN1 with alanines (GYG349–351AAA or HCN1-AAA). HCN1-AAA did not yield functional currents; coexpression of HCN1-AAA with wild-type (WT) HCN1 suppressed normal channel activity in a dominant-negative manner (55.2±3.2%, 68.3±4.3%, 78.7±1.6%, 91.7±0.8%, and 97.9±0.2% current reduction at -140 mV for WT:AAA cRNA ratios of 4:1, 3:1, 2:1, 1:1, and 1:2, respectively) without affecting gating (steady-state activation, activation and deactivation kinetics) or permeation (reversal potential) properties. HCN1-AAA coexpression, however, did not alter the expressed current amplitudes of Kv1.4 and Kv2.1 channels, indicating that its suppressive effect was channel-specific. Statistical analysis reveals that a single HCN channel is composed of 4 monomeric subunits. Interestingly, HCN1-AAA also inhibited HCN2 in a dominant-negative manner with the same efficacy. We conclude that the GYG motif is a critical determinant of ion permeation for HCN channels, and that HCN1 and HCN2 readily coassemble to form heterotetrameric complexes.
Key Words: pacemaker • HCN channels • dominant-negative • signature motif • coassembly
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Introduction |
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Among the key players that underlie cardiac pacing is a hyperpolarization-activated, noninactivating, mixed Na+/K+ current known as If ("f" for funny). Although in the heart, If is most abundant in the sinoatrial node, it is also found at various levels in the atrioventricular node, Purkinje fibers, atria, and ventricles.1 Indeed, upregulated ventricular If in disease states such as heart failure, hypertrophy, and hypertension has been implicated to predispose the associated arrhythmias, albeit somewhat inconsistently.2–5 Despite the recognition of If for over 20 years, the encoding genes, collectively known as the hyperpolarization-activated cyclic-nucleotide–gated (HCN) family, have only been recently cloned.6–8 Four HCN isoforms, namely HCN1 to 4, have been identified; these isoforms have different patterns of gene expression and tissue distribution.1,7–9 By functional inference, it has been proposed that different HCN isoforms coassemble to form heteromeric channel complexes.10,11 Although substantial structural and functional differences are known to exist between HCN and voltage-gated K+ (Kv) channels, the homology they share (for instance, their fundamental building blocks are both monomeric subunits consisting of 6 transmembrane (TM) segments; Figure 1) has led to the speculation that HCN channels are also tetramers like their K+ channel counterparts. However, no direct experimental proof is available.
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Although the pore region of HCN channels also contains the glycine-tyrosine-glycine (GYG) signature motif found in almost all K+-selective channels (Figure 1), pacemaker channels permeate both Na+ and K+. In the present study, we probed the functional importance of the GYG selectivity motif in pacemaker channels by replacing this triplet in HCN1 with alanines (GYG349–351AAA to create HCN1-AAA) using site-directed mutagenesis. Channel constructs were expressed in a heterologous system and characterized electrophysiologically. We found that the GYG motif is indeed a critical determinant for the permeation properties of HCN channels. Coexpression of HCN1-AAA with HCN1 and HCN2 enabled us to probe channel stoichiometry and provide direct evidence that different HCN isoforms can coassemble to form heterotetrameric complexes.
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Materials and Methods |
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Molecular Biology and Heterologous Expression
mHCN1 and mHCN2 (kind gifts of Drs Steven Siegelbaum and Bina Santoro, Columbia University, New York, NY) were subcloned into the pGH expression vector.8 Site-directed mutagenesis was performed using polymerase chain reaction (PCR) with overlapping mutagenic primers as previously described.12 HCN1-AAA was sequenced to ensure that the desired mutations were present. cRNA was transcribed from NheI- and SphI-linearized DNA using T7 RNA polymerase (Promega, Madison, Wis) for HCN1 and HCN2 channels, respectively. Channels were heterologously expressed and studied in Xenopus oocytes. Briefly, stage IV through VI oocytes were surgically removed from female frogs anesthetized by immersion in 1% tricaine (ie, 3-aminobenzoic acid ethyl ester) followed by digestion with 2 mg/mL collagenase in OR-2 containing (in mmol/L) 88 NaCl, 2 KCl, 1 MgCl2, and 5 HEPES (pH 7.6 with NaOH) for 30 to 60 minutes. Isolated oocytes were injected with cRNA (1 ng/nL) as indicated and stored in ND96 solution containing (in mmol/L) 96 NaCl, 2 KCl, 1.8 CaCl2, 1 MgCl2, and 5 HEPES (pH 7.6) supplemented with 50 µg/mL gentamicin, 5 mmol/L pyruvate, and 0.5 mmol/L theophylline for 1 to 4 days before experiments. We found that injection with 50 to 100 ng of total cRNA per cell was sufficient to attain maximal expression, whereas 10 to 25 ng/cell corresponds to the range linearly proportional to the expressed current amplitude (data not shown).
Electrophysiology
Two-electrode voltage-clamp recordings were performed at room temperature (23 to 25°C) using a Warner OC-725C amplifier. Agarose-plugged electrodes (TW120F-6; World Precision Instruments) were pulled using a Sutter P-87 horizontal puller, filled with 3 mol/L KCl, and had final tip resistances of 2 to 4 M
. The recording bath solution contained (in mmol/L) 96 KCl, 2 NaCl, 10 HEPES, and 2 MgCl2 (pH 7.5 with KOH). The currents were digitized at 10 kHz and low-pass filtered at 1 to 2 kHz (-3 dB). Acquisition and analysis of current records were performed using custom-written software.
Experimental Protocols and Data analysis
The steady-state current-voltage (I-V) relationship was determined by plotting the HCN1 currents measured at the end of a 3-second pulse ranging from -150 to 0 mV at 10-mV increments from a holding potential of -30 mV. The voltage dependence of HCN channel activation was assessed by plotting tail currents measured immediately after pulsing to -140 mV as a function of the preceding 3-second test pulse normalized to the maximum tail current recorded. Data were fit to the Boltzmann functions using the Marquardt-Levenberg algorithm in a nonlinear least-squares procedure: m
=1/{1+exp[(Vt-V1/2)/k]}, where Vt is the test potential, V1/2 is the half-point of the relationship, and k=RT/zF is the slope factor.
For reversal potentials (Erev), tail currents were recorded immediately after stepping to a family of test voltages ranging from -100 to +40 mV preceded by a 3-second prepulse to either -140 (cf, Figure 5B) or -20 mV. The difference of tail currents resulting from the 2 prepulse potentials was plotted against the test potentials and fitted with linear regression to obtain Erev. For current kinetics, the time constants for activation (
act) and deactivation (deact) were estimated by fitting macroscopic and tail currents, respectively, with a monoexponential function.
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Data are presented as mean±SEM. Statistical significance was determined using an unpaired Student’s t test with P<0.05 representing significance.
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Results |
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Triple Mutation GYG349–351AAA Rendered HCN1 Channels Nonfunctional
We initially tested the effects of the triple HCN1 mutation GYG349–351AAA (HCN1-AAA) on channel function by individually expressing wild-type (WT) and mutant channel constructs. Figure 2 shows that hyperpolarization of oocytes injected with WT HCN1 cRNA to potentials below -40 mV elicited time-dependent inward currents that reached steady-state current amplitudes after
500 ms. Currents increased in amplitude with progressive hyperpolarization. These properties were consistent with those previously reported for heterologous expression of this HCN isoform.8 In contrast, uninjected oocytes and those injected with HCN1-AAA did not yield measurable currents, indicating that the triple alanine substitution rendered HCN1 completely nonfunctional.
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HCN1-AAA Suppressed the Normal Activity of WT HCN1 in a Dominant-Negative Manner
Previous studies have identified numerous ion channel mutations that are capable of crippling channel activities in a dominant-negative manner when normal and defective subunits coassemble to form multimeric complexes.13–19 We conjectured that, by analogy with K+ channels, HCN1-AAA would exert a dominant-negative effect when combined with WT HCN1 subunits. We tested this hypothesis by coexpressing both WT HCN1 and HCN1-AAA channel constructs. Figure 3 shows that oocytes coinjected with 50 nL WT HCN1 and 50 nL HCN1-AAA cRNA (concentration=1 ng/nL) expressed currents 85.2±1.9% (n=8) smaller than cells injected with 50 nL of WT HCN1 cRNA alone when measured at -140 mV after the same incubation period (P<0.01; Figure 3B). Such quantitative differences existed throughout almost the entire activation range of HCN1 channels as indicated by their corresponding steady-state current-voltage relationships (Figure 3C). These observations demonstrate that HCN1-AAA could suppress the normal activity of WT HCN1 channels in a dominant-negative fashion despite the presence of the same numbers of functional subunits (assuming equal RNA stability and translation efficiencies, as is conventional in previous K+ channel studies).20,21 In contrast, coinjection of 50 nL WT HCN1 cRNA with an equal volume of dH2O yielded current magnitudes not different from the injection of 50 nL WT HCN1 alone (P>0.05), suggesting that the dominant-negative suppressive effects observed with HCN1-AAA were not due to nonspecific mechanisms such as mechanosensitive effects. Indeed, the inhibitory effect of HCN1-AAA was channel-specific because current reductions were not observed when HCN1-AAA was coexpressed with the similar-sized 6 TM Kv1.4 or Kv2.1 channels (provided by Drs G. Tomaselli, Johns Hopkins University, Baltimore, Md, and R. Joho, University of Texas Southern Medical Center, Dallas, Tex, respectively; Figures 4A and 4B; P>0.05). Having confirmed the specificity of our dominant-negative construct, we next studied the effects of varying the ratio of WT HCN1:HCN1-AAA while maintaining the total cRNA injected constant (25 ng was used to prevent saturation of expression; see Materials and Methods). As anticipated from a dominant-negative mechanism, current suppression increased as the proportion of HCN1-AAA increased (55.2±3.2%, 68.3±4.3%, 78.7±1.6%, 91.7±0.8%, and 97.9±0.2% current reduction for WT HCN1:HCN1-AAA ratios of 4:1, 3:1, 2:1, 1:1, and 1:2, respectively; n=9 to 18; Figure 4C).
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Coexpression of the Dominant-Negative Construct HCN1-AAA With WT HCN1 Did Not Alter Normal Gating and Permeation Properties
We then investigated whether coexpression of HCN1-AAA with WT HCN1 affected gating and permeation properties in addition to its dominant-negative suppressive effects on current amplitudes. Figure 5A shows that both the midpoints and slope factors derived from the steady-state activation curves of WT HCN1 alone (V1/2=-76.7±0.8 mV; k=13.3±0.6 mV; n=15) and after suppression by HCN1-AAA (ratio=1:1; V1/2=-77.0±1.7 mV; k=12.3±1.0 mV; n=12) were identical (P>0.05). Tail current-voltage relationships also indicate that whereas whole-cell currents were suppressed by HCN1-AAA, the reversal potential was not changed (WT HCN1 alone=-4.5±1.4 mV, n=8; WT+AAA=-5.25±0.8 mV, n=5; P>0.05; Figures 5B and 5C). Similarly, the time constants for current activation (act) and deactivation (deact), whose distribution was bell-shaped with midpoints comparable to those derived from the corresponding steady-state activation curves, were also unaltered after HCN1-AAA suppression across the entire voltage range studied (P>0.05; Figure 5D). Taken together, our observations indicate that the nonsuppressed currents exhibited normal gating and permeation phenotypes.
HCN1-AAA Suppressed WT HCN2 Currents Without Altering Gating and Permeation
If different HCN isoforms can coassemble to form heteromeric channel complexes, HCN1-AAA should also suppress the activities of WT HCN2 channels in a dominant-negative manner similar to our observations with WT HCN1. Figure 6 shows that this was indeed the case. Currents recorded from oocytes coinjected with 50 nL WT HCN2 and 50 nL HCN1-AAA cRNA were significantly smaller than those expressed in oocytes injected with 50 nL WT HCN2 alone or 50 nL WT HCN2+50 nL dH2O after the same incubation period (Figures 6A through 6C). In fact, the extent of suppression by HCN1-AAA were similar for both WT HCN1 and HCN2 for all other ratios studied (56.5±2.2%, 73.2±3.2%, 82.4±1.5%, and 93.3±1.6% reduction of currents for WT HCN2:HCN1-AAA ratios of 4:1, 3:1, 2:1, and 1:1, respectively; n=6 to 9; Figure 4C; total cRNA injected=25 ng). Taken together, these results indicate that the 2 isoforms were able to coassemble with equivalent efficacy. Similar to HCN1, steady-state activation parameters, reversal potential, and gating kinetics of the nonsuppressed HCN2 currents were not changed by HCN1-AAA coexpression (P>0.05; Figures 6D through 6F).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Our results clearly indicate that the GYG K+-selective channel signature motif is also a critical determinant for ion permeation of HCN channels. Not only did the AAA mutation completely abolish channel function, its coexpression with WT HCN channel subunits also inhibited the expressed currents in a dominant-negative fashion. Such inhibitory effect of HCN1-AAA was channel-specific, as demonstrated by its lack of effects on the similar-sized 6 TM Kv1.4 and Kv2.1 channels. Therefore, it was unlikely that HCN1-AAA coexpression reduced HCN1- and HCN2-encoded currents via nonspecific mechanisms such as competition for protein synthetic or trafficking machinery. HCN1-AAA, however, is likely to exert its suppressive effect by coassembling with WT subunits, because the presence of even just one such dominant-negative monomeric subunit should suffice to render the entire channel complex nonfunctional.13–19 In other words, all monomers present in a channel must be functional in order to form an ion-conducting pore. This notion is supported by the observation that the currents remaining from suppression had gating and permeation properties indistinguishable from those of WT HCN1 or HCN2 expressed alone because functional channels should contain only WT monomers. Because stoichiometric combinations of WT and HCN1-AAA monomers should be stochastic, the probability of any given channel complex containing only functional WT monomers (PWT only) should follow a polynomial distribution and equal the proportion of WT monomers (pWT) available to the nth power (ie, PWT only=pWTn), where n is the number of monomers present in a channel.20 Therefore, PWT only should decrease as the proportion of HCN1-AAA subunits (pAAA) available for coassembling increases. This notion is in complete accordance with our experimental observations. Indeed, using this statistical platform, our data were most consistent with the suppression-pWT relationship predicted from tetramerization of HCN monomers (ie, pWT4; cf, Figure 4). These results provide the first quantitative demonstration that a single HCN channel is made up of 4 subunits. Although not unanticipated based on the structural homology of K+ channels, the tetrameric nature of HCN channels is nevertheless reassuring given their otherwise unusual gating and permeation properties.
Recent experiments involving tandem heterodimers and coexpression of HCN isoforms with different activation kinetics and cAMP sensitivities yield intermediate phenotypes that cannot be explained by the simple addition of individual isoform properties at any population proportional ratio.10,11 These results inferentially suggest that distinct HCN isoforms may heteromultimerize in vivo to form native If currents. The present evidence confirms this interpretation while circumventing a number of potential pitfalls in the "intermediate-phenotype" approach. For instance, coexpression could lead to the competition of certain limiting cellular cofactors (such as a ß subunit22) among different isoforms, thus giving rise to channel properties different from the individual isoforms when expressed alone.10 Tandem dimers could alter properties simply due to the fusion of the C- and N-termini.11 Furthermore, dimeric, trimeric, tetrameric, and even pentameric tandem constructs can form functional "tetrameric" channels by contributing 1 or more of their monomeric repeats for channel assembly, as demonstrated convincingly with Shaker K+ channels.23 Therefore, the intermediate phenotypes observed could result from a mixed population of homomeric channels of both isoforms interacting in some unpredictable manner. In contrast, the ability of HCN1-AAA to cripple also the activity of HCN2, and the observations that the extents of suppression of either WT HCN1 or HCN2 by HCN1-AAA were similar at equivalent WT:AAA ratios when compared, provide complementary experimental evidence that HCN1 and HCN2 can coassemble to form heteromeric complexes without apparent discrimination between isoforms. Our experiments, however, do not allow us to exclude the possibility that HCN1-AAA could block the expression of WT channels by competitively binding to an important limiting factor. Overall, given the complementary approaches and their different associated assumptions, the results from these experiments are mutually reinforcing.
The ability of different HCN isoforms to coassemble with identical efficacies exemplifies the potentially diverse molecular identity of native If, because the currents in different heart regions may be composed of a combination of channel complexes consisting of up to all 4 different isoforms (assuming that the conclusions here can be generalized to HCN3 and HCN4) in various ratios. In any event, our dominant-negative construct, when combined with somatic gene transfer techniques, may provide a useful tool to suppress native If in the sinoatrial node, thereby revealing the physiological contribution of this diastolic current to cardiac pacing. Such a construct can also be used to probe the role of If in ventricular tissue; gene therapy to suppress the upregulated ventricular If found in disease states would logically be expected to inhibit any associated arrhythmias that may occur.2–5
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Acknowledgments |
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We thank Peihong Dong for constructing all channel mutants. This work was supported by a grant from the National Institutes of Health (R01 HL-52768 to E.M.) and a Research Career Development Award from the Cardiac Arrhythmias Research & Education Foundation (to R.A.L.). E.M. holds the Michel Mirowski, MD, Professorship of Cardiology of the Johns Hopkins University.
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Footnotes |
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This manuscript was sent to Harry A. Fozzard, Consulting Editor, for review by expert referees, editorial decision, and final disposition.
Received March 27, 2002; revision received May 17, 2002; accepted May 22, 2002.
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B. Santoro, B. J. Wainger, and S. A. Siegelbaum Regulation of HCN Channel Surface Expression by a Novel C-Terminal Protein-Protein Interaction J. Neurosci., November 24, 2004; 24(47): 10750 - 10762. [Abstract] [Full Text] [PDF]
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S. Y. Tsang, H. Lesso, and R. A. Li Dissecting the Structural and Functional Roles of the S3-S4 Linker of Pacemaker (Hyperpolarization-activated Cyclic Nucleotide-modulated) Channels by Systematic Length Alterations J. Biol. Chem., October 15, 2004; 279(42): 43752 - 43759. [Abstract] [Full Text] [PDF]
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B. Much, C. Wahl-Schott, X. Zong, A. Schneider, L. Baumann, S. Moosmang, A. Ludwig, and M. Biel Role of Subunit Heteromerization and N-Linked Glycosylation in the Formation of Functional Hyperpolarization-activated Cyclic Nucleotide-gated Channels J. Biol. Chem., October 31, 2003; 278(44): 43781 - 43786. [Abstract] [Full Text] [PDF]
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H. Lesso and R. A. Li Helical Secondary Structure of the External S3-S4 Linker of Pacemaker (HCN) Channels Revealed by Site-dependent Perturbations of Activation Phenotype J. Biol. Chem., June 13, 2003; 278(25): 22290 - 22297. [Abstract] [Full Text] [PDF]
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 P. Kirchhof, L. Fabritz, L. Fortmuller, G. P. Matherne, A. Lankford, H. A. Baba, W. Schmitz, G. Breithardt, J. Neumann, and P. Boknik Altered sinus nodal and atrioventricular nodal function in freely moving mice overexpressing the A1 adenosine receptor Am J Physiol Heart Circ Physiol, June 5, 2003; 285(1): H145 - H153. [Abstract] [Full Text] [PDF]
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C. A. Henrikson, T. Xue, P. Dong, D. Sang, E. Marban, and R. A. Li Identification of a Surface Charged Residue in the S3-S4 Linker of the Pacemaker (HCN) Channel That Influences Activation Gating J. Biol. Chem., April 11, 2003; 278(16): 13647 - 13654. [Abstract] [Full Text] [PDF]
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E. Azene, T. Xue, and R. A Li Molecular Basis of the Effect of Potassium on Heterologously Expressed Pacemaker (HCN) Channels J. Physiol., March 1, 2003; 547(2): 349 - 356. [Abstract] [Full Text] [PDF]
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F. Er, R. Larbig, A. Ludwig, M. Biel, F. Hofmann, D. J. Beuckelmann, and U. C. Hoppe Dominant-Negative Suppression of HCN Channels Markedly Reduces the Native Pacemaker Current If and Undermines Spontaneous Beating of Neonatal Cardiomyocytes Circulation, January 28, 2003; 107(3): 485 - 489. [Abstract] [Full Text] [PDF]
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T. Xue and R. A. Li An External Determinant in the S5-P Linker of the Pacemaker (HCN) Channel Identified by Sulfhydryl Modification J. Biol. Chem., November 22, 2002; 277(48): 46233 - 46242. [Abstract] [Full Text] [PDF]
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