Structural basis of inward rectifying potassium channel gating

The last 10 years have seen rapid advances in the understanding of potassium channel function. Since the first inward rectifying (Kir) channels were cloned in 1994, the structural basis of channel function has been significantly elucidated, and determination of the crystal structure of a bacterial K channel (KcsA) in 1998 provided an atomic resolution of the permeation pathway. This review considers recent experimental studies aimed at uncovering the structural basis of Kir channel activity, and the applicability of comparative models based on KcsA to illuminate Kir channel pore structure and opening and closing processes.     

Control of a final gating charge transition by a hydrophobic residue in the S2 segment of a K+ channel voltage sensor

It is now well established that the voltage-sensor domains present in voltage-gated ion channels and some phosphatases operate by transferring several charged residues (gating charges), mainly arginines located in the S4 segment, across the electric field. The conserved phenylalanine F(290) located in the S2 segment of the Shaker K channel is an aromatic residue thought to interact with all the four gating arginines carried by the S4 segment and control their transfer [Tao X, et al. (2010) Science 328:67-73]. In this paper we study the possible interaction of the gating charges with this residue by directly detecting their movement with gating current measurements in 12 F(290) mutants. Most mutations do not significantly alter the first approximately 80-90% of the gating charge transfer nor the kinetics of the gating currents during activation. The effects of the F(290) mutants are (i) the modification of a final activation transition accounting for approximately 10-20% of the total charge, similar to the effect of the ILT mutant [Ledwell JL, et al. (1999) J Gen Physiol 113:389-414] and (ii) the modification of the kinetics of the gating charge movement during deactivation. These effects are well correlated with the hydrophobicity of the substituted residue, showing that a hydrophobic residue at position 290 controls the energy barrier of the final gating transition. Our results suggest that F(290) controls the transfer of R(371), the fourth gating charge, during gating while not affecting the movement of the other three gating arginines.     

Sequential formation of ion pairs during activation of a sodium channel voltage sensor

Electrical signaling in biology depends upon a unique electromechanical transduction process mediated by the S4 segments of voltage-gated ion channels. These transmembrane segments are driven outward by the force of the electric field on positively charged amino acid residues termed “gating charges,” which are positioned at three-residue intervals in the S4 transmembrane segment, and this movement is coupled to opening of the pore. Here, we use the disulfide-locking method to demonstrate sequential ion pair formation between the fourth gating charge in the S4 segment (R4) and two acidic residues in the S2 segment during activation. R4 interacts first with E70 at the intracellular end of the S2 segment and then with D60 near the extracellular end. Analysis with the Rosetta Membrane method reveals the 3-D structures of the gating pore as these ion pairs are formed sequentially to catalyze the S4 transmembrane movement required for voltage-dependent activation. Our results directly demonstrate sequential ion pair formation that is an essential feature of the sliding helix model of voltage sensor function but is not compatible with the other widely discussed gating models.     

Structural basis for gating mechanisms of a eukaryotic P-glycoprotein homolog

P-glycoprotein is an ATP-binding cassette multidrug transporter that actively transports chemically diverse substrates across the lipid bilayer. The precise molecular mechanism underlying transport is not fully understood. Here, we present crystal structures of a eukaryotic P-glycoprotein homolog, CmABCB1 from Cyanidioschyzon merolae, in two forms: unbound at 2.6-Å resolution and bound to a unique allosteric inhibitor at 2.4-Å resolution. The inhibitor clamps the transmembrane helices from the outside, fixing the CmABCB1 structure in an inward-open conformation similar to the unbound structure, confirming that an outward-opening motion is required for ATP hydrolysis cycle. These structures, along with site-directed mutagenesis and transporter activity measurements, reveal the detailed architecture of the transporter, including a gate that opens to extracellular side and two gates that open to intramembranous region and the cytosolic side. We propose that the motion of the nucleotide-binding domain drives those gating apparatuses via two short intracellular helices, IH1 and IH2, and two transmembrane helices, TM2 and TM5.Multidrug transporters of the ATP-binding cassette (ABC) superfamily, such as P-glycoprotein (P-gp; MDR1; ABCB1), MRP1 (ABCC1), and ABCG2 (BCRP), transport a large number of structurally unrelated compounds with molecular weights ranging up to several thousand Daltons (1, 2). These transporters not only play important roles in normal physiology by protecting tissues from various toxic xenobiotics and endogenous metabolites but also contribute to multidrug resistance (MDR) in tumors, a major obstacle to effective chemotherapeutic treatment (1, 3–7). Their functional forms consist of a minimum of four core domains: two transmembrane domains (TMDs) that create the translocation pathway for substrates and two nucleotide-binding domains (NBDs) that bind and hydrolyze ATP to power the transport process (8, 9). These four domains can exist either as two separate polypeptides (half-size) or fused together in a single large polypeptide with an internal duplication (full-size). The crystal structures of mouse and nematode P-gps, as well as their bacterial homologs (10–14), have been determined, and they have provided important insights into the relationships between protein structure and the functional and biochemical characteristics of P-gp. However, the detailed architecture of the TMD machinery and the gating mechanism during the transition between the inward- and outward-open states are poorly understood.Here, we report the structures of a eukaryotic P-gp homolog, unlocked (at 2.6-Å resolution) and locked allosterically with a tailor-made peptide at 2.4-Å resolution. Although CmABCB1 is not a full-length ABC transporter but a half-sized ABC transporter adopting a homodimeric architecture, CmABCB1 showed quite similar functional properties to those of human P-gp (hP-gp). Based on these structures, we propose mechanisms by which the intramembranous entrance gate can take up various substrates from the inner leaflet of the membrane bilayer. Although the entrance and exit gates are assembled from distinct transmembrane helices, and their gating motions are in opposite directions, the mechanisms powered by the dimerization motions of the NBDs enable one gate to close while the other gate simultaneously opens with the aid of two short intracellular helices and two transmembrane helices, which act as a lever arm. This mechanism is totally different from that of solute carrier (SLC) transporters (15–19) and ABC importers (20), in which the intra- and extracellular gating apparatuses are constructed from the same transmembrane helices. Furthermore, a part of the intramembranous entrance gate has another function gating the pathway from the inside of the transporter to the cytosol. The mode of action of the novel inhibitor, which disables the diverging outward motions of the TM helices by clamping them from the outside of the transporter, supports our proposed gating mechanism.     

Disulfide locking a sodium channel voltage sensor reveals ion pair formation during activation

The S4 transmembrane segments of voltage-gated ion channels move outward on depolarization, initiating a conformational change that opens the pore, but the mechanism of S4 movement is unresolved. One structural model predicts sequential formation of ion pairs between the S4 gating charges and negative charges in neighboring S2 and S3 transmembrane segments during gating. Here, we show that paired cysteine substitutions for the third gating charge (R3) in S4 and D60 in S2 of the bacterial sodium channel NaChBac form a disulfide bond during activation, thus “locking” the S4 segment and inducing slow inactivation of the channel. Disulfide locking closely followed the kinetics and voltage dependence of activation and was reversed by hyperpolarization. Activation of D60C:R3C channels is favored compared with single cysteine mutants, and mutant cycle analysis revealed strong free-energy coupling between these residues, further supporting interaction of R3 and D60 during gating. Our results demonstrate voltage-dependent formation of an ion pair during activation of the voltage sensor in real time and suggest that this interaction catalyzes S4 movement and channel activation.     

Dynamic PIP2 interactions with voltage sensor elements contribute to KCNQ2 channel gating

The S4 segment and the S4–S5 linker of voltage-gated potassium (Kv) channels are crucial for voltage sensing. Previous studies on the Shaker and Kv1.2 channels have shown that phosphatidylinositol-4,5-bisphosphate (PIP₂) exerts opposing effects on Kv channels, up-regulating the current amplitude, while decreasing the voltage sensitivity. Interactions between PIP₂ and the S4 segment or the S4–S5 linker in the closed state have been highlighted to explain the effects of PIP₂ on voltage sensitivity. Here, we show that PIP₂ preferentially interacts with the S4–S5 linker in the open-state KCNQ2 (Kv7.2) channel, whereas it contacts the S2–S3 loop in the closed state. These interactions are different from the PIP₂–Shaker and PIP₂–Kv1.2 interactions. Consistently, PIP₂ exerts different effects on KCNQ2 relative to the Shaker and Kv1.2 channels; PIP₂ up-regulates both the current amplitude and voltage sensitivity of the KCNQ2 channel. Disruption of the interaction of PIP₂ with the S4–S5 linker by a single mutation decreases the voltage sensitivity and current amplitude, whereas disruption of the interaction with the S2–S3 loop does not alter voltage sensitivity. These results provide insight into the mechanism of PIP₂ action on KCNQ channels. In the closed state, PIP₂ is anchored at the S2–S3 loop; upon channel activation, PIP₂ interacts with the S4–S5 linker and is involved in channel gating.A series of ion channels, such as inward rectifier K⁺ (Kir) channels, transient receptor potential channels, and voltage-gated channels, are sensitive to the presence of phosphatidylinositol-4,5-bisphosphate (PIP₂) in membranes (1–4). Structural studies on Kir channels (1, 2, 5) demonstrated that PIP₂ directly interacts with the channels. Subsequent studies supported that PIP₂ also interacts directly with voltage-gated potassium (Kv) channels (6–19). Several positive residues that may be critical for PIP₂ activity have been identified (7, 11, 18, 20–24). Previous studies on Kv1.2 and Shaker channels showed that PIP₂ exerts opposing effects on Kv channels, up-regulating the current amplitude, while leading to a decrease in voltage sensitivity (7, 18). The S4 segment and the S4–S5 linker of Kv channels are crucial for voltage sensing. The interactions of PIP₂ with the S4 segments and the S4–S5 linkers of the closed-state Shaker and Kv1.2 channels underlie the loss-of-function effect of PIP₂ on voltage sensitivity (7, 18).The KCNQ (Kv7) family of slowly activated outwardly rectifying potassium channels is one of the Kv channel families that are sensitive to the presence of PIP₂ in the membrane. KCNQ channels have been widely studied because of their important biological and pharmacological functions. Retigabine, a first-in-class K⁺ channel opener used for the treatment of epilepsy, adopts a unique mechanism to enhance the activity of KCNQ channels (25). PIP₂ is important for the functions of KCNQ channels. Reduction of PIP₂ affinity caused by congenic mutations of KCNQ channels is associated with long QT syndrome, suggesting critical physiological implications of PIP₂ on KCNQ channels (23, 26). We reported that PIP₂ also alters the pharmacological selectivity of KCNQ potassium channels (6). Zaydman et al. (27) showed that the coupling of voltage sensing and pore opening in the KCNQ1 channel requires PIP₂ and suggested there is a PIP₂ interaction site at the interface between the voltage-sensing domain (VSD) and the central pore domain (PD). However, the effects and interactions of PIP₂ on KCNQ channels are not well understood.Here, by combining molecular dynamics (MD) simulations, mutagenesis, and electrophysiological determinations, we observed that the effects and interactions of PIP₂ on KCNQ2 are different relative to the Shaker and Kv1.2 channels. PIP₂ up-regulates both the current amplitude and voltage sensitivity of the KCNQ2 channel. PIP₂ preferentially interacts with the S4–S5 linker of the open-state KCNQ2 channel and does not interact with the S4 segment or S4-S5 linker of the closed state. In the closed state, PIP₂ only interacts with the S2–S3 loop. Furthermore, our electrophysiological experiments suggest that disruption of the interaction of PIP₂ with the S4–S5 linker may decrease the voltage sensitivity and current amplitude, whereas disruption of the interaction with the S2–S3 loop only alters the current amplitude of the channel. These results provide insights into the mechanism of PIP₂ action on Kv channels.     

Gating charge interactions with the S1 segment during activation of a Na+ channel voltage sensor

Voltage-gated Na(+) channels initiate action potentials during electrical signaling in excitable cells. Opening and closing of the pore of voltage-gated ion channels are mechanically linked to voltage-driven outward movement of the positively charged S4 transmembrane segment in their voltage sensors. Disulfide locking of cysteine residues substituted for the outermost T0 and R1 gating-charge positions and a conserved negative charge (E43) at the extracellular end of the S1 segment of the bacterial Na(+) channel NaChBac detects molecular interactions that stabilize the resting state of the voltage sensor and define its conformation. Upon depolarization, the more inward gating charges R2 and R3 engage in these molecular interactions as the S4 segment moves outward to its intermediate and activated states. The R4 gating charge does not disulfide-lock with E43, suggesting an outer limit to its transmembrane movement. These molecular interactions reveal how the S4 gating charges are stabilized in the resting state and how their outward movement is catalyzed by interaction with negatively charged residues to effect pore opening and initiate electrical signaling.     

Gating transitions in the selectivity filter region of a sodium channel are coupled to the domain IV voltage sensor

Voltage-dependent ion channels are crucial for generation and propagation of electrical activity in biological systems. The primary mechanism for voltage transduction in these proteins involves the movement of a voltage-sensing domain (D), which opens a gate located on the cytoplasmic side. A distinct conformational change in the selectivity filter near the extracellular side has been implicated in slow inactivation gating, which is important for spike frequency adaptation in neural circuits. However, it remains an open question whether gating transitions in the selectivity filter region are also actuated by voltage sensors. Here, we examine conformational coupling between each of the four voltage sensors and the outer pore of a eukaryotic voltage-dependent sodium channel. The voltage sensors of these sodium channels are not structurally symmetric and exhibit functional specialization. To track the conformational rearrangements of individual voltage-sensing domains, we recorded domain-specific gating pore currents. Our data show that, of the four voltage sensors, only the domain IV voltage sensor is coupled to the conformation of the selectivity filter region of the sodium channel. Trapping the outer pore in a particular conformation with a high-affinity toxin or disulphide crossbridge impedes the return of this voltage sensor to its resting conformation. Our findings directly establish that, in addition to the canonical electromechanical coupling between voltage sensor and inner pore gates of a sodium channel, gating transitions in the selectivity filter region are also coupled to the movement of a voltage sensor. Furthermore, our results also imply that the voltage sensor of domain IV is unique in this linkage and in the ability to initiate slow inactivation in sodium channels.     

Voltage-dependent K+ channel gating and voltage sensor toxin sensitivity depend on the mechanical state of the lipid membrane

Voltage-dependent K⁺ (Kv) channels underlie action potentials through gating conformational changes that are driven by membrane voltage. In this study of the paddle chimera Kv channel, we demonstrate that the rate of channel opening, the voltage dependence of the open probability, and the maximum achievable open probability depend on the lipid membrane environment. The activity of the voltage sensor toxin VsTx1, which interferes with voltage-dependent gating by partitioning into the membrane and binding to the channel, also depends on the membrane. Membrane environmental factors that influence channel function are divisible into two general categories: lipid compositional and mechanical state. The mechanical state can have a surprisingly large effect on the function of a voltage-dependent K⁺ channel, including its pharmacological interaction with voltage sensor toxins. The dependence of VSTx1 activity on the mechanical state of the membrane leads us to hypothesize that voltage sensor toxins exert their effect by perturbing the interaction forces that exist between the channel and the membrane.     

Detergent extraction of a presumptive gating component from the voltage-dependent sodium channel.

A physiologically characterized radiolabeled neurotoxin complex obtained from venom of the scorpion Leiurus quinquestriatus has been used to identify detergent-solubilized presumptive sodium channel components in sucrose gradients. This toxin-binding component is found in extracts prepared from three sources of excitable membrane but appears to be absent from similar extracts prepared from nonexcitable membrane or from Torpedo californica membrane. Procedures that destroy the physiological activity of the Leiurus neurotoxin lead to a corresponding loss of toxin binding to the putative sodium channel component. The major component recognized by the Leiurus toxin sediments at 6.5 S. Scatchard analysis of quantitative binding experiments carried out in sucrose gradients shows approximately linear plots and indicates that the toxin recognizes a relatively small number of sites with a dissociation constant near 10 nM. Once formed, the channel element--toxin complex is quite stable. Experiments show diphasic dissociation kinetics with half-times near 70 hr and greater than 200 hr.     

Structural basis for alcohol modulation of a pentameric ligand-gated ion channel

Despite its long history of use and abuse in human culture, the molecular basis for alcohol action in the brain is poorly understood. The recent determination of the atomic-scale structure of GLIC, a prokaryotic member of the pentameric ligand-gated ion channel (pLGIC) family, provides a unique opportunity to characterize the structural basis for modulation of these channels, many of which are alcohol targets in brain. We observed that GLIC recapitulates bimodal modulation by n-alcohols, similar to some eukaryotic pLGICs: methanol and ethanol weakly potentiated proton-activated currents in GLIC, whereas n-alcohols larger than ethanol inhibited them. Mapping of residues important to alcohol modulation of ionotropic receptors for glycine, γ-aminobutyric acid, and acetylcholine onto GLIC revealed their proximity to transmembrane cavities that may accommodate one or more alcohol molecules. Site-directed mutations in the pore-lining M2 helix allowed the identification of four residues that influence alcohol potentiation, with the direction of their effects reflecting α-helical structure. At one of the potentiation-enhancing residues, decreased side chain volume converted GLIC into a highly ethanol-sensitive channel, comparable to its eukaryotic relatives. Covalent labeling of M2 positions with an alcohol analog, a methanethiosulfonate reagent, further implicated residues at the extracellular end of the helix in alcohol binding. Molecular dynamics simulations elucidated the structural consequences of a potentiation-enhancing mutation and suggested a structural mechanism for alcohol potentiation via interaction with a transmembrane cavity previously termed the "linking tunnel." These results provide a unique structural model for independent potentiating and inhibitory interactions of n-alcohols with a pLGIC family member.     

Modulating the voltage sensor of a cardiac potassium channel shows antiarrhythmic effects

Cardiac arrhythmias are the most common cause of sudden cardiac death worldwide. Lengthening the ventricular action potential duration (APD), either congenitally or via pathologic or pharmacologic means, predisposes to a life-threatening ventricular arrhythmia, Torsade de Pointes. I_Ks (KCNQ1+KCNE1), a slowly activating K⁺ current, plays a role in action potential repolarization. In this study, we screened a chemical library in silico by docking compounds to the voltage-sensing domain (VSD) of the I_Ks channel. Here, we show that C28 specifically shifted I_Ks VSD activation in ventricle to more negative voltages and reversed the drug-induced lengthening of APD. At the same dosage, C28 did not cause significant changes of the normal APD in either ventricle or atrium. This study provides evidence in support of a computational prediction of I_Ks VSD activation as a potential therapeutic approach for all forms of APD prolongation. This outcome could expand the therapeutic efficacy of a myriad of currently approved drugs that may trigger arrhythmias.

I_Ks (KCNQ1+KCNE1), a slowly activating delayer rectifier in the heart, is important in controlling cardiac action potential duration (APD) and adaptation of heart rate in various physiological conditions (1). The I_Ks potassium channel has slow activation kinetics, and the activation terminates cardiac action potentials (APs) (2). This channel is formed by the voltage-gated potassium (K_V) channel subunit KCNQ1 and the regulatory subunit KCNE1. The association of KCNE1 drastically alters the phenotype of the channel, including a shift of voltage dependence of activation to more positive voltages, a slower activation time course, a changed ion selectivity, and different responses to drugs and modulators (3–6). Similar to other K_V channels, KCNQ1 has six transmembrane segments, S1 to S6, in which S1 to S4 form the voltage-sensing domain (VSD), while S5 and S6 form the pore domain (PD); four KCNQ1 subunits comprise the KCNQ1 channel (7, 8). KCNQ1 and I_Ks channels are activated by voltage. The VSD in response to membrane depolarization changes conformation, triggered by the movements of the S4 segment that contains positively charged residues (9–15). This conformational change alters the interactions between the VSD and the pore, known as the VSD–pore coupling, to induce pore opening (12–15).The ventricular APD depends on the balance of outward and inward currents flowing at plateau potentials. The outward currents include the delayed rectifiers I_Kr and I_Ks, while the inward currents include a persistent sodium current (I_NaP) (16). Specific mutations in any of these channel proteins that cause a reduction in outward current or increase in inward current are associated with congenital long QT syndrome (LQTS). The QT interval is the time between the initial depolarization of the ventricle until the time to full repolarization. LQTS is a condition in which the APD is abnormally prolonged, predisposing the afflicted patients to a lethal cardiac arrhythmia called Torsades de Pointes (TdP) (17). In fact, mutations in multiple genes that alter the function of various ion channels have been associated with LQTS (18). There is also a much more prevalent problem called acquired LQTS (aLQTS) that is most often associated with off target effects of drugs. Many drugs are marketed with a QT prolongation warning, and the drug concentrations that can be used therapeutically are limited by this potentially lethal side effect. Some effective drugs have been removed from the market (19) because of QT prolongation, and others are abandoned before clinical trials even began. Therefore, aLQTS is costly for the pharmaceutical industry both in drug development (to avoid this side effect) and when it results in removal from the market of compounds that have effectively treated other diseases (20, 21). At present, the I_Kr (HERG) potassium channel (20) and the phosphoinositide 3-kinase (PI3K) (22, 23) have been identified as the most prominent off targets of these drugs for the association with aLQTS.We hypothesized that in LQTS, the normal heart function can be restored and QT prolongation prevented by compensating for the change in net current from any of the channels produced by the myocyte; all that is required is that a reasonable facsimile of normal net current flow be restored. We applied this approach to aLQTS based on a computational study to show that a shift of voltage-dependent activation of I_Ks to more negative voltages would increase I_Ks during ventricular APs; this increase of I_Ks would revert drug-induced APD prolongation to normal. More importantly, a change in I_Ks voltage-dependent activation might affect the normal APD to a much smaller degree because of its slow activation kinetics in healthy ventricular myocytes, thereby posing minimal risk of cardiac toxicity on its own. To apply this approach experimentally, we needed a compound that could specifically shift the voltage dependence of I_Ks activation. Previous studies showed that the benzodiazepine R-L3 (24) and polyunsaturated fatty acids (25) activate I_Ks channels, while more recently, rottlerin was shown to act similarly to R-L3 (26). However, the effects of these compounds on I_Ks are complex, likely through binding to more than one site in the channel protein instead of simply acting on voltage-dependent activation (24, 27, 28). In addition, these compounds showed poor specificity for I_Ks, also affecting other ion channels in the heart (27, 29, 30), which make these compounds unsuitable to test our hypothesis. Recently, we have identified CP1 as an activator for I_Ks, which mimics the membrane lipid phosphatidylinositol 4,5-bisphosphate (PIP₂) to mediate the VSD–pore coupling (13, 31). CP1 enhances I_Ks primarily by increasing current amplitude with some shift of voltage dependence of activation, which is not suitable for our test either.In this study, we identified a compound, C28, using an approach that combines in silico and experimental screening, that interacts with the KCNQ1 VSD and shifts voltage dependence of VSD activation to more negative voltages. C28 increases both exogenously expressed I_Ks and the current in native cardiac myocytes. As predicted by computational modeling, C28 can prevent or reverse the drug-induced APD prolongation back to normal while having a minimal effect on the control APD at the same concentration in healthy cardiac myocytes. This study demonstrates that the KCNQ1 VSD can be used as a drug target for developing a therapy for LQTS, and C28 identified in this study may be used as a lead for this development. Furthermore, our results provide support for the use of docking computations based on ion channel structure and cellular physiology, in combination with functional studies based on molecular mechanisms, as an effective approach for rational drug design.     

Mapping the receptor site for alpha-scorpion toxins on a Na+ channel voltage sensor

The α-scorpions toxins bind to the resting state of Na(+) channels and inhibit fast inactivation by interaction with a receptor site formed by domains I and IV. Mutants T1560A, F1610A, and E1613A in domain IV had lower affinities for Leiurus quinquestriatus hebraeus toxin II (LqhII), and mutant E1613R had ~73-fold lower affinity. Toxin dissociation was accelerated by depolarization and increased by these mutations, whereas association rates at negative membrane potentials were not changed. These results indicate that Thr1560 in the S1-S2 loop, Phe1610 in the S3 segment, and Glu1613 in the S3-S4 loop in domain IV participate in toxin binding. T393A in the SS2-S6 loop in domain I also had lower affinity for LqhII, indicating that this extracellular loop may form a secondary component of the receptor site. Analysis with the Rosetta-Membrane algorithm resulted in a model of LqhII binding to the voltage sensor in a resting state, in which amino acid residues in an extracellular cleft formed by the S1-S2 and S3-S4 loops in domain IV interact with two faces of the wedge-shaped LqhII molecule. The conserved gating charges in the S4 segment are in an inward position and form ion pairs with negatively charged amino acid residues in the S2 and S3 segments of the voltage sensor. This model defines the structure of the resting state of a voltage sensor of Na(+) channels and reveals its mode of interaction with a gating modifier toxin.     

Evidence for developmental changes in sodium channel inactivation gating and sodium channel block by phenytoin in rat cardiac myocytes.

The voltage-dependent properties of the voltage-activated sodium channel were studied in neonatal (1-2-day-old) and adult rat ventricular cardiac myocytes using the whole-cell variation of the patch-clamp technique (16 degrees C, [Na]i = 15 mM, [Na]o = 25 mM). The voltage dependence of the sodium conductance-membrane potential relation was similar in both neonatal and adult myocytes except for a difference in slope; the adult sodium conductance-membrane potential relation was slightly more steep. Neonatal cells also differed from adult cells by demonstrating a more negative voltage midpoint of their sodium availability curve, a slower rate of recovery from inactivation at hyperpolarized potentials, and a greater extent of slow inactivation development compared with adult cells. Phenytoin (40 microM) reduced the sodium current in a tonic and use-dependent manner in both adult and neonatal myocytes. However, phenytoin (40 microM) produced significantly more tonic block at negative holding potentials (e.g., -140 mV) in neonatal myocytes (22 +/- 5% [mean +/- SEM], n = 14) than in adult myocytes (10 +/- 2%, n = 11) (p less than 0.05). The amplitudes of use-dependent block obtained during trains of 1-second pulses to -20 mV were also significantly greater in neonatal myocytes than in adult myocytes when the diastolic interval was varied over a range of 0.1-1.5 seconds (p less than 0.05). Definition of the time courses of block development at -20 mV indicated that phenytoin had a slightly higher affinity for inactivated sodium channels in neonatal cells. In addition, the time constant of recovery from use-dependent block by phenytoin was found to be significantly longer in neonatal cells than in adult cells at membrane potentials between -160 and -100 mV (p less than 0.001). The marked differences in phenytoin effect on cardiac sodium channels in neonatal versus adult rat cardiac myocytes suggest that there may be significant developmental changes in the sodium channel blocking effects of class I antiarrhythmic drugs in cardiac tissue.     

Structural interactions of a voltage sensor toxin with lipid membranes

Protein toxins from tarantula venom alter the activity of diverse ion channel proteins, including voltage, stretch, and ligand-activated cation channels. Although tarantula toxins have been shown to partition into membranes, and the membrane is thought to play an important role in their activity, the structural interactions between these toxins and lipid membranes are poorly understood. Here, we use solid-state NMR and neutron diffraction to investigate the interactions between a voltage sensor toxin (VSTx1) and lipid membranes, with the goal of localizing the toxin in the membrane and determining its influence on membrane structure. Our results demonstrate that VSTx1 localizes to the headgroup region of lipid membranes and produces a thinning of the bilayer. The toxin orients such that many basic residues are in the aqueous phase, all three Trp residues adopt interfacial positions, and several hydrophobic residues are within the membrane interior. One remarkable feature of this preferred orientation is that the surface of the toxin that mediates binding to voltage sensors is ideally positioned within the lipid bilayer to favor complex formation between the toxin and the voltage sensor.Protein toxins from venomous organisms have been invaluable tools for studying the ion channel proteins they target. For example, in the case of voltage-activated potassium (Kv) channels, pore-blocking scorpion toxins were used to identify the pore-forming region of the channel (1, 2), and gating modifier tarantula toxins that bind to S1–S4 voltage-sensing domains have helped to identify structural motifs that move at the protein–lipid interface (3–5). In many instances, these toxin–channel interactions are highly specific, allowing them to be used in target validation and drug development (6–8).Tarantula toxins are a particularly interesting class of protein toxins that have been found to target all three families of voltage-activated cation channels (3, 9–12), stretch-activated cation channels (13–15), as well as ligand-gated ion channels as diverse as acid-sensing ion channels (ASIC) (16–21) and transient receptor potential (TRP) channels (22, 23). The tarantula toxins targeting these ion channels belong to the inhibitor cystine knot (ICK) family of venom toxins that are stabilized by three disulfide bonds at the core of the molecule (16, 17, 24–31). Although conventional tarantula toxins vary in length from 30 to 40 aa and contain one ICK motif, the recently discovered double-knot toxin (DkTx) that specifically targets TRPV1 channels contains two separable lobes, each containing its own ICK motif (22, 23).One unifying feature of all tarantula toxins studied thus far is that they act on ion channels by modifying the gating properties of the channel. The best studied of these are the tarantula toxins targeting voltage-activated cation channels, where the toxins bind to the S3b–S4 voltage sensor paddle motif (5, 32–36), a helix-turn-helix motif within S1–S4 voltage-sensing domains that moves in response to changes in membrane voltage (37–41). Toxins binding to S3b–S4 motifs can influence voltage sensor activation, opening and closing of the pore, or the process of inactivation (4, 5, 36, 42–46). The tarantula toxin PcTx1 can promote opening of ASIC channels at neutral pH (16, 18), and DkTx opens TRPV1 in the absence of other stimuli (22, 23), suggesting that these toxin stabilize open states of their target channels.For many of these tarantula toxins, the lipid membrane plays a key role in the mechanism of inhibition. Strong membrane partitioning has been demonstrated for a range of toxins targeting S1–S4 domains in voltage-activated channels (27, 44, 47–50), and for GsMTx4 (14, 50), a tarantula toxin that inhibits opening of stretch-activated cation channels in astrocytes, as well as the cloned stretch-activated Piezo1 channel (13, 15). In experiments on stretch-activated channels, both the d- and l-enantiomers of GsMTx4 are active (14, 50), implying that the toxin may not bind directly to the channel. In addition, both forms of the toxin alter the conductance and lifetimes of gramicidin channels (14), suggesting that the toxin inhibits stretch-activated channels by perturbing the interface between the membrane and the channel. In the case of Kv channels, the S1–S4 domains are embedded in the lipid bilayer and interact intimately with lipids (48, 51, 52) and modification in the lipid composition can dramatically alter gating of the channel (48, 53–56). In one study on the gating of the Kv2.1/Kv1.2 paddle chimera (53), the tarantula toxin VSTx1 was proposed to inhibit Kv channels by modifying the forces acting between the channel and the membrane. Although these studies implicate a key role for the membrane in the activity of Kv and stretch-activated channels, and for the action of tarantula toxins, the influence of the toxin on membrane structure and dynamics have not been directly examined. The goal of the present study was to localize a tarantula toxin in membranes using structural approaches and to investigate the influence of the toxin on the structure of the lipid bilayer.     

Structural basis of action for a human ether-a-go-go-related gene 1 potassium channel activator

Activation of human ether-a-go-go-related gene 1 (hERG1) K(+) channels mediates cardiac action potential repolarization. Drugs that activate hERG1 channels represent a mechanism-based approach for the treatment of long QT syndrome, a disorder of cardiac repolarization associated with ventricular arrhythmia and sudden death. Here, we characterize the mechanisms of action and the molecular determinants for binding of RPR260243 [(3R,4R)-4-[3-(6-methoxy-quinolin-4-yl)-3-oxo-propyl]-1-[3-(2,3,5-trifluoro-phenyl)-prop-2-ynyl]-piperidine-3-carboxylic acid] (RPR), a recently discovered hERG1 channel activator. Channels were heterologously expressed in Xenopus laevis oocytes, and currents were measured by using the two-microelectrode voltage-clamp technique. RPR induced a concentration-dependent slowing in the rate of channel deactivation and enhanced current magnitude by shifting the voltage dependence of inactivation to more positive potentials. This mechanism was confirmed by demonstrating that RPR slowed the rate of deactivation, but did not increase current magnitude of inactivation-deficient mutant channels. The effects of RPR on hERG1 kinetics and magnitude could be simulated by reducing three rate constants in a Markov model of channel gating. Point mutations of specific residues located in the S4-S5 linker or cytoplasmic ends of the S5 and S6 domains greatly attenuated or ablated the effects of 3 microM RPR on deactivation (five residues), inactivation (one residue), or both gating mechanisms (four residues). These findings define a putative binding site for RPR and confirm the importance of an interaction between the S4-S5 linker and the S6 domain in electromechanical coupling of voltage-gated K(+) channels.     

Reversed voltage-dependent gating of a bacterial sodium channel with proline substitutions in the S6 transmembrane segment

Members of the voltage-gated-like ion channel superfamily have a conserved pore structure. Transmembrane helices that line the pore (M2 or S6) are thought to gate it at the cytoplasmic end by bending at a hinge glycine residue. Proline residues favor bending of alpha-helices, and substitution of proline for this glycine (G219) dramatically stabilizes the open state of a bacterial Na(+) channel NaChBac. Here we have probed S6 pore-lining residues of NaChBac by proline mutagenesis. Five of 15 proline-substitution mutants yielded depolarization-activated Na(+) channels, but only G219P channels have strongly negatively shifted voltage dependence of activation, demonstrating specificity for bending at G219 for depolarization-activated gating. Remarkably, three proline-substitution mutations on the same face of S6 as G219 yielded channels that activated upon hyperpolarization and inactivated very slowly. Studies of L226P showed that hyperpolarization to -147 mV gives half-maximal activation, 123 mV more negative than WT. Analysis of combination mutations and studies of block by the local anesthetic etidocaine favored the conclusion that hyperpolarization-activated gating results from opening of the cytoplasmic gate formed by S6 helices. Substitution of multiple amino acids for L226 indicated that hyperpolarization-activated gating was correlated with a high propensity for bending, whereas depolarization-activated gating was favored by a low propensity for bending. Our results further define the dominant role of bending of S6 in determining not only the voltage dependence but also the polarity of voltage-dependent gating. Native hyperpolarization-activated gating of hyperpolarization- and cyclic nucleotide-gated (HCN) channels in animals and KAT channels in plants may involve bending at analogous S6 amino acid residues.     

Molecular basis of multistep voltage activation in plant two-pore channel 1

Voltage-gated ion channels confer excitability to biological membranes, initiating and propagating electrical signals across large distances on short timescales. Membrane excitation requires channels that respond to changes in electric field and couple the transmembrane voltage to gating of a central pore. To address the mechanism of this process in a voltage-gated ion channel, we determined structures of the plant two-pore channel 1 at different stages along its activation coordinate. These high-resolution structures of activation intermediates, when compared with the resting-state structure, portray a mechanism in which the voltage-sensing domain undergoes dilation and in-membrane plane rotation about the gating charge–bearing helix, followed by charge translocation across the charge transfer seal. These structures, in concert with patch-clamp electrophysiology, show that residues in the pore mouth sense inhibitory Ca²⁺ and are allosterically coupled to the voltage sensor. These conformational changes provide insight into the mechanism of voltage-sensor domain activation in which activation occurs vectorially over a series of elementary steps.

Voltage-gated ion channels (VGICs) use voltage-sensing domains (VSDs) to sense changes in electrical potential across biological membranes (1, 2). VSDs are composed of a four-helix bundle, in which one helix carries charged residues that move in response to changes in transmembrane electric field (3, 4). VSDs usually adopt a “resting state” when the membrane is at “resting potential”: ∼ −80 mV for animal and ∼ −150 mV for plant plasma membranes (5). In comparison, much lower resting membrane voltages are set for intracellular endo-membranes: ∼ −30 mV across the plant vacuole (6) and mammalian lysosome (7). As the membrane potential vanishes during depolarization, so does the downward electrostatic force on the cationic side chains causing them to relax toward the outside of the membrane across a hydrophobic constriction site (HCS) or hydrophobic seal (8). This conformational change is conveyed to the central pore, formed by four pore domains in a quasi-fourfold arrangement, which dilates to allow the diffusion of ions down their electrochemical gradients. The exact nature of the conformational change in VSDs has been the subject of decades of biophysical investigation (9–12), though to this date, only a few structural examples exist of voltage sensors in resting or multiple conformations (13–18).Two-pore channels (TPCs) are defined by their two tandem Shaker-like cassette subunits in a single polypeptide chain, which dimerize to form a C2-symmetric channel with four subunits and 24 (4 × 6) transmembrane helices (19–21). There are three TPC channels, TPC1, 2, and 3, each with different voltage or ligand gating and ion selectivity. Among the voltage-gated TPCs (all except lipid-gated TPC2), only the second VSD (VSD2) is electrically active (18, 22–24), while VSD1 is insensitive to voltage changes and is likely static under all changes in potential.In plants, the vacuole comprises up to 90% of the plant cell volume and provides for a dynamic storage organelle that, in addition to metabolites, is a repository for ions including Ca²⁺. TPC1 channels confer excitability to this intracellular organelle (25) and, unlike other TPCs, are calcium regulated: external Ca²⁺ (in the vacuolar lumen) inhibits the channel by binding to multiple luminal sites, while cytosolic Ca²⁺ is required to open the channel by binding to EF hands, although the exact mechanism by which this activation occurs is unknown (22). These electrical properties allowed our group and Youxing Jiang’s group to determine the first structure of an electrically resting VGIC by cocrystallizing the channel with 1 mM Ca²⁺, which maintains the VSD in a resting configuration at 0 mV potential (16, 22).Previously (17), we used a gain-of-function mutant of AtTPC1 with three luminal Ca²⁺-binding acidic residues on VSD2 neutralized (D240N/D454N/E528N) termed AtTPC1_DDE (abbreviated here as DDE) to visualize channel activation at the level of atomic structure, but we were unable to sufficiently resolve details of the electrically active VSD2 due to structural heterogeneity. In addition, the intracellular activation gate remained closed. We now present multiple structures of intermediately activated states of AtTPC1 determined by extensive image processing. In order to visualize such states, we modulated the channel’s luminal Ca²⁺ sensitivity using a well-studied gain-of-function single-point mutant, D454N (fou2), and also the triple mutant DDE for comparison. fou2 is known to desensitize the channel to inhibitory, external (luminal) calcium ions (26, 27). Mutations in D454 and closely related luminal Ca²⁺-binding carboxyls to alanine D240A, D454A, E528A (termed AtTPC1ΔCa_i) were previously shown to effectively attenuate the Ca²⁺-induced shift of the voltage activation threshold of AtTPC1 to depolarizing potentials at high luminal Ca²⁺ (28).The fou2 and DDE mutations lie in the coordination sphere of the inhibitory Ca²⁺ site on the luminal side of the VSD2–pore interface formed by D454, D240, and E528. The D454N mutation in the fou2 channel enhances the defense capacity of plants against fungal or herbivore attack due to increased production of the wounding hormone jasmonate (29). These effects on plant performance and defense are probably due to short circuiting of the vacuolar membrane (26, 27, 30) in which TPC1 has increased open probability at resting potential. Compared to wild-type (WT) TPC1, the activation threshold in the fou2 channel is shifted to more negatively polarized potentials and also has significantly lower sensitivity to inhibitory Ca²⁺ in addition to exhibiting faster activation kinetics than its WT counterpart that was originally named the “slow vacuolar” (SV) channel due to its slow conductance onset (20, 21, 30). Therefore, D454N confers more than just reduced sensitivity to external Ca²⁺ but intrinsic hyperactivity as well. Our structures of these AtTPC1 mutants attempt to explain how the voltage sensor functions during electrical activation and how exactly luminal Ca²⁺ affects this process.