Helical secondary structure of the external S3-S4 linker of pacemaker (HCN) channels revealed by site-dependent perturbations of activation phenotype

If, encoded by the hyperpolarization-activated cyclic nucleotide-modulated channel family (HCN1-4), contributes significantly to neuronal and cardiac pacing. Recently, we reported that the S3-S4 residue Glu-235 of HCN1 influences activation by acting as a surface charge. However, it is uncertain whether other residues of the external S3-S4 linker are also involved in gating. Furthermore, the secondary conformation of the linker is not known. Here we probed the structural and functional role of the HCN1 S3-S4 linker by introducing systematic mutations into the entire linker (defined as 229-237) and studying their effects. We found that the mutations K230A (-62.2 +/- 3.4 mV versus -72.2 +/- 1.7 mV of wild type (WT)), G231A (-64.4 +/- 1.3 mV), M232A (V(1/2) = -63.1 +/- 1.1 mV), and E235G (-65.4 +/- 1.5 mV) produced depolarizing activation shifts. Although E229A and M232A decelerated gating kinetics (<13- and 3-fold, respectively), K230A and G231A accelerated both activation and deactivation (< approximately 2-3-fold). D233A, S234A, V236A, and Y237A channels exhibited WT properties (p > 0.05). Shortening the linker (EVY235-237deltadeltadelta) caused depolarizing activation shift and slowed kinetics that could not be explained by removing the charge at position 235 alone. Secondary structural predictions by the modeling algorithms SSpro2 and PROF, along with refinements by our experimental data, suggest that part of the S3-S4 linker conforms a helical structure with the functionally important residues Met-232, Glu-235, and Gly-231 (|deltadeltaG|>1 kcal/mol) clustered on one side.     

The analysis of desensitizing CNGA1 channels reveals molecular interactions essential for normal gating

The pore region of cyclic nucleotide–gated (CNG) channels acts as the channel gate. Therefore, events occurring in the cyclic nucleotide–binding (CNB) domain must be coupled to the movements of the pore walls. When Glu363 in the pore region, Leu356 and Thr355 in the P helix, and Phe380 in the upper portion of the S6 helix are mutated into an alanine, gating is impaired: mutant channels E363A, L356A, T355A, and F380A desensitize in the presence of a constant cGMP concentration, contrary to what can be observed in wild-type (WT) CNGA1 channels. Similarly to C-type inactivation of K⁺ channels, desensitization in these mutant channels is associated with rearrangements of residues in the outer vestibule. In the desensitized state, Thr364 residues in different subunits become closer and Pro366 becomes more accessible to extracellular reagents. Desensitization is also observed in the mutant channel L356C, but not in the double-mutant channel L356C+F380C. Mutant channels L356F and F380K did not express, but cGMP-gated currents with a normal gating were observed in the double-mutant channels L356F+F380L and L356D+F380K. Experiments with tandem constructs with L356C, F380C, and L356C+F380C and WT channels indicate that the interaction between Leu356 and Phe380 is within the same subunit. These results show that Leu356 forms a hydrophobic interaction with Phe380, coupling the P helix with S6, whereas Glu363 could interact with Thr355, coupling the pore wall to the P helix. These interactions are essential for normal gating and underlie the transduction between the CNB domain and the pore.     

Structural and functional determinants in the S5-P region of HCN-encoded pacemaker channels revealed by cysteine-scanning substitutions

Hyperpolarization-activated cyclic nucleotide-modulated (HCN) channels are responsible for the membrane pacemaker current that underlies the spontaneous generation of bioelectrical rhythms. However, their structure-function relationship is poorly understood. Previously, we identified several pore residues that influence HCN gating properties and proposed a pore-to-gate mechanism. Here, we systematically introduced cysteine-scanning substitutions into the descending portion of the P loop (residues 339-345) of HCN1-R (where R is resistance to sulfhydryl-reactive agents) channels, in which all endogenous cysteines except C303 have been removed or replaced. F339C, K340C, A341C, M342C, S343C, and M345C did not produce functional currents. Interestingly, the loss of function phenotype of F339C could be rescued by the reducing agent dithiothreitol (DTT). H344C but not HCN1-R and DTT-treated F339C channels were sensitive to blockade by divalent Cd(2+) (current with 100 microM Cd(2+)/control current at -140 mV = 67.6 +/- 2.9%, 109.3 +/- 3.1%, and 103.8 +/- 1.7%, respectively). Externally applied methanethiosulfate ethylammonium, a covalent sulfhydryl-reactive compound, irreversibly modified H344C by reducing the current at -140 mV (to 43.7 +/- 6.5%), causing a hyperpolarizing steady-state activation shift (change in half-activation voltage: approximately 6 mV) and decelerated gating kinetics (by up to 3-fold). Based on these results, we conclude that pore residues 339-345 are important determinants of the structure-function properties of HCN channels and that the side chain of H344 is externally accessible.     

An external determinant in the S5-P linker of the pacemaker (HCN) channel identified by sulfhydryl modification

Hyperpolarization-activated, cyclic nucleotide-gated (HCN) channels underlie spontaneous rhythmic activities in the heart and brain. Sulfhydryl modification of ion channels is a proven approach for studying their structure-function relationships; here we examined the effects of the hydrophilic sulfhydryl-modifying agents methanethiosulfonate ethylammonium (MTSEA(+)) and methanethiosulfonate ethylsulfonate (MTSES(-)) on wild-type (WT) and engineered HCN1 channels. External application of MTSEA(+) to WT channels irreversibly reduced whole-cell currents (I(MTSEA)/I(Control) = 42 +/- 2%), slowed activation and deactivation kinetics ( approximately 7- and approximately 3-fold at -140 and -20 mV, respectively), and produced hyperpolarizing shifts of steady-state activation (V(12)((MTSEA)) = -125.8 +/- 9.0 mV versus V(12)((Control)) = -76.4 +/- 1.6 mV). Sequence inspection revealed the presence of five endogenous cysteines in the transmembrane domains of HCN1: three are putatively close to the extracellular milieu (Cys(303), Cys(318), and Cys(347) in the S5, S5-P, and P segments, respectively), whereas the remaining two are likely to be cytoplasmic or buried. To identify the molecular constituent(s) responsible for the effects of MTSEA(+), we mutated the three "external" cysteines individually to serine. C303S did not yield measurable currents. Whereas C347S channels remained sensitive to MTSEA(+), C318S was not modified (I(MTSEA)/I(Control) = 101 +/- 2%, V(12)((MTSEA)) = -78.4 +/- 1.1 mV, and V(12)((Control)) = -79.8 +/- 2.3 mV). Likewise, WT (but not C318S) channels were sensitive to MTSES(-). Despite their opposite charges, MTSES(-) produced changes directionally similar to those effected by MTSEA(+) (I(MTSES)/I(Control) = 22 +/- 1.6% and V(12)((MTSES)) = -145.9 +/- 4.9 mV). We conclude that S5-P Cys(318) of HCN1 is externally accessible and that the external pore vestibule and activation gating of HCN channels are allosterically coupled.     

Uncoupling of gating charge movement and closure of the ion pore during recovery from inactivation in the Kv1.5 channel

Both wild-type (WT) and nonconducting W472F mutant (NCM) Kv1.5 channels are able to conduct Na(+) in their inactivated states when K(+) is absent. Replacement of K(+) with Na(+) or NMG(+) allows rapid and complete inactivation in both WT and W472F mutant channels upon depolarization, and on return to negative potentials, transition of inactivated channels to closed-inactivated states is the first step in the recovery of the channels from inactivation. The time constant for immobilized gating charge recovery at -100 mV was 11.1 +/- 0.4 ms (n = 10) and increased to 19.0 +/- 1.6 ms (n = 3) when NMG(+)(o) was replaced by Na(+)(o). However, the decay of the Na(+) tail currents through inactivated channels at -100 mV had a time constant of 129 +/- 26 ms (n = 18), much slower than the time required for gating charge recovery. Further experiments revealed that the voltage-dependence of gating charge recovery and of the decay of Na(+) tail currents did not match over a 60 mV range of repolarization potentials. A faster recovery of gating charge than pore closure was also observed in WT Kv1.5 channels. These results provide evidence that the recovery of the gating elements is uncoupled from that of the pore in Na(+)-conducting inactivated channels. The dissociation of the gating charge movements and the pore closure could also be observed in the presence of symmetrical Na(+) but not symmetrical Cs(+). This difference probably stems from the difference in the respective abilities of the two ions to limit inactivation to the P-type state or prevent it altogether.     

Identification of the Molecular Site of Ivabradine Binding to HCN4 Channels

Ivabradine is a specific heart rate-reducing agent approved as a treatment of chronic stable angina. Its mode of action involves a selective and specific block of HCN channels, the molecular components of sinoatrial "funny" (f)-channels. Different studies suggest that the binding site of ivabradine is located in the inner vestibule of HCN channels, but the molecular details of ivabradine binding are unknown. We thus sought to investigate by mutagenesis and in silico analysis which residues of the HCN4 channel, the HCN isoform expressed in the sinoatrial node, are involved in the binding of ivabradine. Using homology modeling, we verified the presence of an inner cavity below the channel pore and identified residues lining the cavity; these residues were replaced with alanine (or valine) either alone or in combination, and WT and mutant channels were expressed in HEK293 cells. Comparison of the block efficiency of mutant vs WT channels, measured by patch-clamp, revealed that residues Y506, F509 and I510 are involved in ivabradine binding. For each mutant channel, docking simulations correctly explain the reduced block efficiency in terms of proportionally reduced affinity for ivabradine binding. In summary our study shows that ivabradine occupies a cavity below the channel pore, and identifies specific residues facing this cavity that interact and stabilize the ivabradine molecule. This study provides an interpretation of known properties of f/HCN4 channel block by ivabradine such as the “open channel block”, the current-dependence of block and the property of "trapping" of drug molecules in the closed configuration.     

Compromised E-cadherin adhesion and epithelial barrier function with activation of G protein-coupled receptors is rescued by Y-to-F mutations in beta-catenin

Activation of the type 1 histamine (H1) or the type 2 protease-activated (PAR-2) G protein-coupled receptors interrupts E-cadherin adhesion and decreases the transepithelial resistance (TER) of epithelium. Several reports suggest that cadherin adhesive function depends on the association of cadherin with beta-catenin and that this association is regulated by phosphorylation of tyrosines in beta-catenin. We tested the hypothesis that loss of cadherin adhesion and compromise of TER on activation of the H1 or PAR-2 receptor is due to phosphorylation of tyrosines in beta-catenin. L cells were stably transfected to express E-cadherin (L-E-cad cells) and H1 (L-H1-E-cad cells). L cells and Madin-Darby canine kidney (MDCK) cells constitutively express PAR-2. Stably transfected L-E-cad, L-H1-E-cad, and MDCK cells were also stably transfected with FLAG-tagged wild-type (WT) or mutant beta-catenin, converting tyrosine 142, 489, or 654 to the nonphosphorylatable mimetic, phenylalanine (WT, Y142F, Y489F, or Y654F). Activation of H1 or PAR-2 interrupted adhesion to an immobilized E-cadherin-Fc fusion protein of L-H1-E-cad, L-E-cad, and MDCK cells expressing WT or Y142F beta-catenin but did not interrupt adhesion of L-H1-E-cad, L-E-cad, and MDCK cells expressing the Y489F or Y654F mutant beta-catenins. PAR-2 activation decreased the TER of monolayers of MDCK cells expressing WT or Y142F beta-catenin 40-45%. However, PAR-2 activation did not decrease the TER of monolayers of MDCK cells expressing Y489F or Y654F beta-catenin. The protein tyrosine phosphatase PTP1B binds to the cadherin cytoplasmic domain and dephosphorylates beta-catenin. Inhibition of PTP1B interrupted adhesion to E-cadherin-Fc of MDCK cells expressing WT beta-catenin but did not affect the adhesion of MDCK cells expressing Y489F or Y654F beta-catenin. Similarly, inhibition of PTP1B compromised the TER of MDCK cells expressing WT beta-catenin but did not affect the TER of MDCK cells expressing Y489F or Y654F beta-catenin. We conclude that phosphorylation of tyrosines 489 and 654 in beta-catenin is a necessary step in the process by which G protein-coupled H1 and PAR-2 receptors interrupt E-cadherin adhesion. We also conclude that activation of PAR-2 has no effect on the TER without first interrupting E-cadherin adhesion.     

Inner activation gate in S6 contributes to the state-dependent binding of cAMP in full-length HCN2 channel

Recently, applications of the patch-clamp fluorometry (PCF) technique in studies of cyclic nucleotide-gated (CNG) and hyperpolarization-activated, cyclic nucleotide-regulated (HCN) channels have provided direct evidence for the long-held notion that ligands preferably bind to and stabilize these channels in an open state. This state-dependent ligand-channel interaction involves contributions from not only the ligand-binding domain but also other discrete structural elements within the channel protein. This insight led us to investigate whether the pore of the HCN channel plays a role in the ligand-whole channel interaction. We used three well-characterized HCN channel blockers to probe the ion-conducting passage. The PCF technique was used to simultaneously monitor channel activity and cAMP binding. Two ionic blockers, Cs(+) and Mg(2+), effectively block channel conductance but have no obvious effect on cAMP binding. Surprisingly, ZD7288, an open channel blocker specific for HCN channels, significantly reduces the activity-dependent increase in cAMP binding. Independent biochemical assays exclude any nonspecific interaction between ZD7288 and isolated cAMP-binding domain. Because ZD7228 interacts with the inner pore region, where the activation gate is presumably located, we did an alanine scanning of the intracellular end of S6, from T426 to A435. Mutations of three residues, T426, M430, and H434, which are located at regular intervals on the S6 α-helix, enhance cAMP binding. In contrast, mutations of two residues in close proximity, F431A and I432A, dampen the response. Our results demonstrate that movements of the structural elements near the activation gate directly affect ligand binding affinity, which is a simple mechanistic explanation that could be applied to the interpretation of ligand gating in general.     

State-Dependent Accessibility of the P-S6 Linker of Pacemaker (HCN) Channels Supports a Dynamic Pore-to-Gate Coupling Model

The hyperpolarization-activated cyclic nucleotide-modulated channel gene family (HCN1-4) encodes the membrane depolarizing current that underlies pacemaking. Although the topology of HCN resembles K_v channels, much less is known about their structure-function correlation. Previously, we identified several pore residues in the S5-P linker and P-loop that are externally accessible and/or influence HCN gating, and proposed an evolutionarily conserved pore-to-gate mechanism. Here we sought dynamic evidence by assessing the functional consequences of Cys-scanning substitutions in the unexplored P-S6 linker (residues 352–359), the HCN1-R background (that is, resistant to sulfhydryl-reactive agents). None of A352C, Q353C, A354C, P355C, V356C, S357C, M358C, or S359C produced functional currents; the loss-of-function of Q353C, A354C, S357C, and M358C could be rescued by the reducing agent dithiothreitol. Q353C, A354C, and S357C, but not M358C and HCN1-R, were sensitive to Cd²⁺ blockade (IC₅₀ = 3–12 μM vs. >1 mM). External application of the positively charged covalent sulfhydryl modifier MTSET irreversibly reduced I _−140mV of Q353C and A354C to 27.9 ± 3.4% and 58.2 ± 13.1% of the control, respectively, and caused significant steady-state activation shifts (∆V _1/2 = –21.1 ± 1.6 for Q353C and −10.0 ± 2.9 mV for A354C). Interestingly, MTSET reactivity was also state dependent. MTSET, however, affected neither S357C nor M358C, indicating site specificity. Collectively, we have identified novel P-S6 residues whose extracellular accessibility was sterically and state dependent and have provided the first functional evidence consistent with a dynamic HCN pore-to-gate model.     

Amino acid substitution at position 99 affects the rate of CRP subunit exchange

We investigated the characteristics of CRP having amino acid substitutions at position 99. Analysis of amino acid residue proximity to cAMP in molecular dynamics (MD) simulations of the CRP:(cAMP)(2) complex [García, A. E., and Harman, J. G. (1996) Protein Sci. 5, 62-71] showed repositioning of tyrosine 99 (Y99) to interact with the equatorial exocyclic oxygen atom of cAMP. To test the role of Y99 in cAMP-mediated CRP activation, Y99 was substituted with alanine (A) or phenylalanine (F). Cells that contained the WT or mutant forms of CRP induced beta-galactosidase in the presence of cAMP. Purified WT, Y99A, and Y99F CRP showed only a 3- to 4-fold difference in cAMP affinity. There were no apparent differences between the three forms of CRP in cAMP binding cooperativity, in CRP:(cAMP)(1) complex binding to lacP DNA, in the formation of CRP:cAMP:RNAP complexes at lacP, or in CRP efficacy in mediating lacP activity in vitro. The apo-form of Y99A CRP was more sensitive to protease than the apo-form of either WT CRP or Y99F CRP. Whereas the WT or Y99F CRP:(cAMP)(1) complexes were cleaved by protease at hinge-region peptide bonds, the Y99A CRP:(cAMP)(1) complex was cleaved at peptide bonds located at the subunit interface. The rates of subunit exchange for Y99A CRP, both in the apo-form and in a 1:1 complex with cAMP, were significantly greater than that measured for WT CRP. The results of this study show that tyrosine 99 contributes significant structural stability to the CRP dimer, specifically in stabilizing subunit association.     

P-Loop Residues Critical for Selectivity in K+ Channels Fail to Confer Selectivity to Rabbit HCN4 Channels

HCN channels are thought to be structurally similar to K_v channels, but show much lower selectivity for K⁺. The ∼3.3 Å selectivity filter of K⁺ channels is formed by the pore-lining sequence XT(V/I)GYG, with X usually T, and is held stable by key residues in the P-loop. Differences in the P-loop sequence of HCN channels (eg. the pore-lining sequence L₄₇₈C₄₇₉IGYG) suggest these residues could account for differences in selectivity between these channel families. Despite being expressed, L478T/C479T HCN4 channels did not produce current. Since threonine in the second position is highly conserved in K⁺ channels, we also studied C479T channels. Based on permeability ratios (P_X/P_K), C479T HCN4 channels (K⁺(1)>Rb⁺(0.85)>Cs⁺(0.59)>Li⁺(0.50)≥Na⁺(0.49)) were less selective than WT rabbit HCN4 (K⁺(1)>Rb⁺(0.48)>Cs⁺(0.31)≥Na⁺(0.29)>Li⁺(0.03)), indicating that the TIGYG sequence is insufficient to confer K⁺ selectivity to HCN channels. C479T HCN4 channels had an increased permeability to large organic cations than WT HCN4 channels, as well as increased unitary K⁺ conductance, and altered channel gating. Collectively, these results suggest that HCN4 channels have larger pores than K⁺ channels and replacement of the cysteine at position 479 with threonine further increases pore size. Furthermore, selected mutations in other regions linked previously to pore stability in K⁺ channels (ie. S475D, S475E and F471W/K472W) were also unable to confer K⁺ selectivity to C479T HCN4 channels. Our findings establish the presence of the TIGYG pore-lining sequence does not confer K⁺ selectivity to rabbit HCN4 channels, and suggests that differences in selectivity of HCN4 versus K⁺ channels originate from differences outside the P-loop region.     

TEA(+)-sensitive KCNQ1 constructs reveal pore-independent access to KCNE1 in assembled I(Ks) channels

I(Ks), a slowly activating delayed rectifier K(+) current through channels formed by the assembly of two subunits KCNQ1 (KvLQT1) and KCNE1 (minK), contributes to the control of the cardiac action potential duration. Coassembly of the two subunits is essential in producing the characteristic and physiologically critical kinetics of assembled channels, but it is not yet clear where or how these subunits interact. Previous investigations of external access to the KCNE1 protein in assembled I(Ks) channels relied on occlusion of the pore by extracellular application of TEA(+), despite the very low TEA(+) sensitivity (estimated EC(50) > 100 mM) of channels encoded by coassembly of wild-type KCNQ1 with the wild type (WT) or a series of cysteine-mutated KCNE1 constructs. We have engineered a high affinity TEA(+) binding site into the h-KCNQ1 channel by either a single (V319Y) or double (K318I, V319Y) mutation, and retested it for pore-delimited access to specific sites on coassembled KCNE1 subunits. Coexpression of either KCNQ1 construct with WT KCNE1 in Chinese hamster ovary cells does not alter the TEA(+) sensitivity of the homomeric channels (IC(50) approximately 0.4 mM [TEA(+)](out)), providing evidence that KCNE1 coassembly does not markedly alter the structure of the outer pore of the KCNQ1 channel. Coexpression of a cysteine-substituted KCNE1 (F54C) with V319Y significantly increases the sensitivity of channels to external Cd(2+), but neither the extent of nor the kinetics of the onset of (or the recovery from) Cd(2+) block was affected by [TEA(+)](o) at 10x the IC(50) for channel block. These data strongly suggest that access of Cd(2+) to the cysteine-mutated site on KCNE1 is independent of pore occlusion caused by TEA(+) binding to the outer region of the KCNE1/V319Y pore, and that KCNE1 does not reside within the pore region of the assembled channels.     

Inactivation gating of Kv7.1 channels does not involve concerted cooperative subunit interactions

Inactivation is an intrinsic property of numerous voltage-gated K⁺ (Kv) channels and can occur by N-type or/and C-type mechanisms. N-type inactivation is a fast, voltage independent process, coupled to activation, with each inactivation particle of a tetrameric channel acting independently. In N-type inactivation, a single inactivation particle is necessary and sufficient to occlude the pore. C-type inactivation is a slower process, involving the outermost region of the pore and is mediated by a concerted, highly cooperative interaction between all four subunits. Inactivation of Kv7.1 channels does not exhibit the hallmarks of N- and C-type inactivation. Inactivation of WT Kv7.1 channels can be revealed by hooked tail currents that reflects the recovery from a fast and voltage-independent inactivation process. However, several Kv7.1 mutants such as the pore mutant L273F generate an additional voltage-dependent slow inactivation. The subunit interactions during this slow inactivation gating remain unexplored. The goal of the present study was to study the nature of subunit interactions along Kv7.1 inactivation gating, using concatenated tetrameric Kv7.1 channel and introducing sequentially into each of the four subunits the slow inactivating pore mutation L273F. Incorporating an incremental number of inactivating mutant subunits did not affect the inactivation kinetics but slowed down the recovery kinetics from inactivation. Results indicate that Kv7.1 inactivation gating is not compatible with a concerted cooperative process. Instead, adding an inactivating subunit L273F into the Kv7.1 tetramer incrementally stabilizes the inactivated state, which suggests that like for activation gating, Kv7.1 slow inactivation gating is not a concerted process.     

MiRP1 modulates HCN2 channel expression and gating in cardiac myocytes

MinK-related protein (MiRP1 or KCNE2) interacts with the hyperpolarization-activated, cyclic nucleotide-gated (HCN) family of pacemaker channels to alter channel gating in heterologous expression systems. Given the high expression levels of MiRP1 and HCN subunits in the cardiac sinoatrial node and the contribution of pacemaker channel function to impulse initiation in that tissue, such an interaction could be of considerable physiological significance. However, the functional evidence for MiRP1/HCN interactions in heterologous expression studies has been accompanied by inconsistencies between studies in terms of the specific effects on channel function. To evaluate the effect of MiRP1 on HCN expression and function in a physiological context, we used an adenovirus approach to overexpress a hemagglutinin (HA)-tagged MiRP1 (HAMiRP1) and HCN2 in neonatal rat ventricular myocytes, a cell type that expresses both MiRP1 and HCN2 message at low levels. HA-MiRP1 co-expression with HCN2 resulted in a 4-fold increase in maximal conductance of pacemaker currents compared with HCN2 expression alone. HCN2 activation and deactivation kinetics also changed, being significantly more rapid for voltages between -60 and -95 mV when HA-MiRP1 was co-expressed with HCN2. However, the voltage dependence of activation was not affected. Co-immunoprecipitation experiments demonstrated that expressed HA-MiRP1 and HCN2, as well as endogenous MiRP1 and HCN2, co-assemble in ventricular myocytes. The results indicate that MiRP1 acts as a beta subunit for HCN2 pacemaker channel subunits and alters channel gating at physiologically relevant voltages in cardiac cells.     

High affinity HERG K(+) channel blockade by the antiarrhythmic agent dronedarone: resistance to mutations of the S6 residues Y652 and F656

Pharmacological inhibition of human-ether-a-go-go-related gene (HERG) K(+) channels by structurally and therapeutically diverse drugs is associated with the 'acquired' form of long QT syndrome and with potentially lethal cardiac arrhythmias. Two aromatic amino-acid residues (Y652 and F656) on the inner (S6) helices are considered to be key constituents of a high affinity drug binding site within the HERG channel pore cavity. Using wild-type (WT) and mutant HERG channels expressed in mammalian cell lines, we have investigated HERG channel current (I(HERG)) blockade at 37+/-1 degrees C by dronedarone (DRONED), a non-iodinated analogue of the Class III antiarrhythmic agent amiodarone (AMIOD). Under our conditions WT I(HERG) tails, measured at -40 mV following activating pulses to +30 mV, were blocked with IC(50) values of approximately 59 and 70 nM for DRONED and AMIOD, respectively. I(HERG) inhibition by DRONED was contingent upon channel gating, with block developing rapidly on membrane depolarization, but with no preference for activated over inactivated channels. High external [K(+)] (94 mM) reduced the potency of I(HERG) inhibition by both DRONED and AMIOD. Strikingly, mutagenesis to alanine of the S6 residue F656 (F656A) failed to eliminate blockade by both DRONED and AMIOD, whilst Y652A had comparatively little effect on DRONED but some effect on AMIOD. These findings demonstrate that high affinity drug blockade of I(HERG) can occur without a strong dependence on the Y652 and F656 aromatic amino-acid residues.     

Investigating the putative glycine hinge in Shaker potassium channel

The crystal structure of an open potassium channel reveals a kink in the inner helix that lines the pore (Jiang, Y.X., A. Lee, J.Y. Chen, M. Cadene, B.T. Chait, and R. MacKinnon. 2002. Nature 417:523-526). The putative hinge point is a highly conserved glycine residue. We examined the role of the homologous residue (Gly466) in the S6 transmembrane segment of Shaker potassium channels. The nonfunctional alanine mutant G466A will assemble, albeit poorly, with wild-type (WT) subunits, suppressing functional expression. To test if this glycine residue is critical for activation gating, we did a glycine scan along the S6 segment in the background of G466A. Although all of these double mutants lack the higher-level glycosylation that is characteristic of mature Shaker channels, one (G466A/V467G) is able to generate voltage-dependent potassium current. Surface biotinylation shows that functional and nonfunctional constructs containing G466A express at comparable levels in the plasma membrane. Compared with WT channels, the shifted-glycine mutant has impairments in voltage-dependent channel opening, including a right-shifted activation curve and a decreased rate of activation. The double mutant has relatively normal open-channel properties, except for a decreased affinity for intracellular blockers, a consequence of the loss of the side chain of Val467. Control experiments with the double mutants M440A/G466A and G466A/V467A suggest that the flexibility provided by Gly466 is more important for channel function than its small size. Our results support roles for Gly466 both in biogenesis of the channel and as a hinge in activation gating.     

Tuning of EAG K+ channel inactivation: Molecular determinants of amplification by mutations and a small molecule

Ether-à-go-go (EAG) and EAG-related gene (ERG) K(+) channels are close homologues but differ markedly in their gating properties. ERG1 channels are characterized by rapid and extensive C-type inactivation, whereas mammalian EAG1 channels were previously considered noninactivating. Here, we show that human EAG1 channels exhibit an intrinsic voltage-dependent slow inactivation that is markedly enhanced in rate and extent by 1-10 μM 3-nitro-N-(4-phenoxyphenyl) benzamide, or ICA105574 (ICA). This compound was previously reported to have the opposite effect on ERG1 channels, causing an increase in current magnitude by inhibition of C-type inactivation. The voltage dependence of 2 μM ICA-induced inhibition of EAG1 current was half-maximal at -73 mV, 62 mV negative to the half-point for channel activation. This finding suggests that current inhibition by the drug is mediated by enhanced inactivation and not open-channel block, where the voltage half-points for current inhibition and channel activation are predicted to overlap, as we demonstrate for clofilium and astemizole. The mutation Y464A in the S6 segment also induced inactivation of EAG1, with a time course and voltage dependence similar to that caused by 2 μM ICA. Several Markov models were investigated to describe gating effects induced by multiple concentrations of the drug and the Y464A mutation. Models with the smallest fit error required both closed- and open-state inactivation. Unlike typical C-type inactivation, the rate of Y464A- and ICA-induced inactivation was not decreased by external tetraethylammonium or elevated [K(+)](e). EAG1 channel inactivation introduced by Y464A was prevented by additional mutation of a nearby residue located in the S5 segment (F359A) or pore helix (L434A), suggesting a tripartite molecular model where interactions between single residues in S5, S6, and the pore helix modulate inactivation of EAG1 channels.     

NMR structures of the second transmembrane domain of the human glycine receptor alpha(1) subunit: model of pore architecture and channel gating

Glycine receptors (GlyR) are the primary inhibitory receptors in the spinal cord and belong to a superfamily of ligand-gated ion channels (LGICs) that are extremely sensitive to low-affinity neurological agents such as general anesthetics and alcohols. The high-resolution pore architecture and the gating mechanism of this superfamily, however, remain unclear. The pore-lining second transmembrane (TM2) segments of the GlyR alpha(1) subunit are unique in that they form functional homopentameric channels with conductance characteristics nearly identical to those of an authentic receptor (Opella, S. J., J. Gesell, A. R. Valente, F. M. Marassi, M. Oblatt-Montal, W. Sun, A. F. Montiel, and M. Montal. 1997. Chemtracts Biochem. Mol. Biol. 10:153-174). Using NMR and circular dichroism (CD), we determined the high-resolution structures of the TM2 segment of human alpha(1) GlyR and an anesthetic-insensitive mutant (S267Y) in dodecyl phosphocholine (DPC) and sodium dodecyl sulfate (SDS) micelles. The NMR structures showed right-handed alpha-helices without kinks. A well-defined hydrophilic path, composed of side chains of G2', T6', T10', Q14', and S18', runs along the helical surfaces at an angle approximately 10-20 degrees relative to the long axis of the helices. The side-chain arrangement of the NMR-derived structures and the energy minimization of a homopentameric TM2 channel in a fully hydrated DMPC membrane using large-scale computation suggest a model of pore architecture in which simultaneous tilting movements of entire TM2 helices by a mere 10 degrees may be sufficient to account for the channel gating. The model also suggests that additional residues accessible from within the pore include L3', T7', T13', and G17'. A similar pore architecture and gating mechanism may apply to other channels in the same superfamily, including GABA(A), nACh, and 5-HT(3) receptors.     

Integrated allosteric model of voltage gating of HCN channels

Hyperpolarization-activated (pacemaker) channels are dually gated by negative voltage and intracellular cAMP. Kinetics of native cardiac f-channels are not compatible with HH gating, and require closed/open multistate models. We verified that members of the HCN channel family (mHCN1, hHCN2, hHCN4) also have properties not complying with HH gating, such as sigmoidal activation and deactivation, activation deviating from fixed power of an exponential, removal of activation "delay" by preconditioning hyperpolarization. Previous work on native channels has indicated that the shifting action of cAMP on the open probability (Po) curve can be accounted for by an allosteric model, whereby cAMP binds more favorably to open than closed channels. We therefore asked whether not only cAMP-dependent, but also voltage-dependent gating of hyperpolarization-activated channels could be explained by an allosteric model. We hypothesized that HCN channels are tetramers and that each subunit comprises a voltage sensor moving between "reluctant" and "willing" states, whereas voltage sensors are independently gated by voltage, channel closed/open transitions occur allosterically. These hypotheses led to a multistate scheme comprising five open and five closed channel states. We estimated model rate constants by fitting first activation delay curves and single exponential time constant curves, and then individual activation/deactivation traces. By simply using different sets of rate constants, the model accounts for qualitative and quantitative aspects of voltage gating of all three HCN isoforms investigated, and allows an interpretation of the different kinetic properties of different isoforms. For example, faster kinetics of HCN1 relative to HCN2/HCN4 are attributable to higher HCN1 voltage sensors' rates and looser voltage-independent interactions between subunits in closed/open transitions. It also accounts for experimental evidence that reduction of sensors' positive charge leads to negative voltage shifts of Po curve, with little change of curve slope. HCN voltage gating thus involves two processes: voltage sensor gating and allosteric opening/closing.     

The Th2 lymphoproliferation developing in LatY136F mutant mice triggers polyclonal B cell activation and systemic autoimmunity

Lat(Y136F) knock-in mice harbor a point mutation in Tyr(136) of the linker for activation of T cells and show accumulation of Th2 effector cells and IgG1 and IgE hypergammaglobulinemia. B cell activation is not a direct effect of the mutation on B cells since in the absence of T cells, mutant B cells do not show an activated phenotype. After adoptive transfer of linker for activation of T cell mutant T cells into wild-type, T cell-deficient recipients, recipient B cells become activated. We show in vivo and in vitro that the Lat(Y136F) mutation promotes T cell-dependent B cell activation leading to germinal center, memory, and plasma cell formation even in an MHC class II-independent manner. All the plasma and memory B cell populations found in physiological T cell-dependent B cell responses are found. Characterization of the abundant plasmablasts found in secondary lymphoid organs of Lat(Y136F) mice revealed the presence of a previously uncharacterized CD93-expressing subpopulation, whose presence was confirmed in wild-type mice after immunization. In Lat(Y136F) mice, B cell activation was polyclonal and not Ag-driven because the increase in serum IgG1 and IgE concentrations involved Abs and autoantibodies with different specificities equally. Although the noncomplement-fixing IgG1 and IgE are the only isotypes significantly increased in Lat(Y136F) serum, we observed early-onset systemic autoimmunity with nephritis showing IgE autoantibody deposits and severe proteinuria. These results show that Th2 cells developing in Lat(Y136F) mice can trigger polyclonal B cell activation and thereby lead to systemic autoimmune disease.