Kv Channel S1-S2 Linker Working as a Binding Site of Human β-Defensin 2 for Channel Activation Modulation

Among the three extracellular domains of the tetrameric voltage-gated K⁺ (Kv) channels consisting of six membrane-spanning helical segments named S1–S6, the functional role of the S1-S2 linker still remains unclear because of the lack of a peptide ligand. In this study, the Kv1.3 channel S1-S2 linker was reported as a novel receptor site for human β-defensin 2 (hBD2). hBD2 shifts the conductance-voltage relationship curve of the human Kv1.3 channel in a positive direction by nearly 10.5 mV and increases the activation time constant for the channel. Unlike classical gating modifiers of toxin peptides from animal venoms, which generally bind to the Kv channel S3-S4 linker, hBD2 only targets residues in both the N and C termini of the S1-S2 linker to influence channel gating and inhibit channel currents. The increment and decrement of the basic residue number in a positively charged S4 sensor of Kv1.3 channel yields conductance-voltage relationship curves in the positive direction by ∼31.2 mV and 2–4 mV, which suggests that positively charged hBD2 is anchored in the channel S1-S2 linker and is modulating channel activation through electrostatic repulsion with an adjacent S4 helix. Together, these findings reveal a novel peptide ligand that binds with the Kv channel S1-S2 linker to modulate channel activation. These findings also highlight the functional importance of the Kv channel S1-S2 linker in ligand recognition and modification of channel activation.     

Systemic Administration of Substance P Recovers Beta Amyloid-Induced Cognitive Deficits in Rat: Involvement of Kv Potassium Channels

Reduced levels of Substance P (SP), an endogenous neuropeptide endowed with neuroprotective and anti-apoptotic properties, have been found in brain and spinal fluid of Alzheimer''s disease (AD) patients. Potassium (K⁺) channel dysfunction is implicated in AD development and the amyloid-β (Aβ)-induced up-regulation of voltage-gated potassium channel subunits could be considered a significant step in Aβ brain toxicity. The aim of this study was to evaluate whether SP could reduce, in vivo, Aβ-induced overexpression of Kv subunits. Rats were intracerebroventricularly infused with amyloid-β 25–35 (Aβ_25–35, 20 µg) peptide. SP (50 µg/Kg, i.p.) was daily administered, for 7 days starting from the day of the surgery. Here we demonstrate that the Aβ infused rats showed impairment in cognitive performances in the Morris water maze task 4 weeks after Aβ_25–35 infusion and that this impairing effect was prevented by SP administration.Kv1.4, Kv2.1 and Kv4.2 subunit levels were quantified in hippocampus and in cerebral cortex by Western blot analysis and immunofluorescence. Interestingly, SP reduced Kv1.4 levels overexpressed by Aβ, both in hippocampus and cerebral cortex.Our findings provide in vivo evidence for a neuroprotective activity of systemic administration of SP in a rat model of AD and suggest a possible mechanism underlying this effect.     

Affinity for phosphatidylinositol 4,5-bisphosphate determines muscarinic agonist sensitivity of Kv7 K+ channels

Kv7 K⁺-channel subunits differ in their apparent affinity for PIP₂ and are differentially expressed in nerve, muscle, and epithelia in accord with their physiological roles in those tissues. To investigate how PIP₂ affinity affects the response to physiological stimuli such as receptor stimulation, we exposed homomeric and heteromeric Kv7.2, 7.3, and 7.4 channels to a range of concentrations of the muscarinic receptor agonist oxotremorine-M (oxo-M) in a heterologous expression system. Activation of M₁ receptors by oxo-M leads to PIP₂ depletion through G_q and phospholipase C (PLC). Chinese hamster ovary cells were transiently transfected with Kv7 subunits and M₁ receptors and studied under perforated-patch voltage clamp. For Kv7.2/7.3 heteromers, the EC₅₀ for current suppression was 0.44 ± 0.08 µM, and the maximal inhibition (Inhib_max) was 74 ± 3% (n = 5–7). When tonic PIP₂ abundance was increased by overexpression of PIP 5-kinase, the EC₅₀ was shifted threefold to the right (1.2 ± 0.1 µM), but without a significant change in Inhib_max (73 ± 4%, n = 5). To investigate the muscarinic sensitivity of Kv7.3 homomers, we used the A315T pore mutant (Kv7.3^T) that increases whole-cell currents by 30-fold without any change in apparent PIP₂ affinity. Kv7.3^T currents had a slightly right-shifted EC₅₀ as compared with Kv7.2/7.3 heteromers (1.0 ± 0.8 µM) and a strongly reduced Inhib_max (39 ± 3%). In contrast, the dose–response curve of homomeric Kv7.4 channels was shifted considerably to the left (66 ± 8 nM), and Inhib_max was slightly increased (81 ± 6%, n = 3–4). We then studied several Kv7.2 mutants with altered apparent affinities for PIP₂ by coexpressing them with Kv7.3^T subunits to boost current amplitudes. For the lower affinity (Kv7.2 (R463Q)/Kv7.3^T) or higher affinity (Kv7.2 (R463E)/Kv7.3^T) channels, the EC₅₀ and Inhib_max were similar to Kv7.4 or Kv7.3^T homomers (0.12 ± 0.08 µM and 79 ± 6% [n = 3–4] and 0.58 ± 0.07 µM and 27 ± 3% [n = 3–4], respectively). The very low-affinity Kv7.2 (R452E, R459E, and R461E) triple mutant was also coexpressed with Kv7.3^T. The resulting heteromer displayed a very low EC₅₀ for inhibition (32 ± 8 nM) and a slightly increased Inhib_max (83 ± 3%, n = 3–4). We then constructed a cellular model that incorporates PLC activation by oxo-M, PIP₂ hydrolysis, PIP₂ binding to Kv7-channel subunits, and K⁺ current through Kv7 tetramers. We were able to fully reproduce our data and extract a consistent set of PIP₂ affinities.     

Two Separate Interfaces between the Voltage Sensor and Pore Are Required for the Function of Voltage-Dependent K+ Channels

Voltage-dependent K⁺ (Kv) channels gate open in response to the membrane voltage. To further our understanding of how cell membrane voltage regulates the opening of a Kv channel, we have studied the protein interfaces that attach the voltage-sensor domains to the pore. In the crystal structure, three physical interfaces exist. Only two of these consist of amino acids that are co-evolved across the interface between voltage sensor and pore according to statistical coupling analysis of 360 Kv channel sequences. A first co-evolved interface is formed by the S4-S5 linkers (one from each of four voltage sensors), which form a cuff surrounding the S6-lined pore opening at the intracellular surface. The crystal structure and published mutational studies support the hypothesis that the S4-S5 linkers convert voltage-sensor motions directly into gate opening and closing. A second co-evolved interface forms a small contact surface between S1 of the voltage sensor and the pore helix near the extracellular surface. We demonstrate through mutagenesis that this interface is necessary for the function and/or structure of two different Kv channels. This second interface is well positioned to act as a second anchor point between the voltage sensor and the pore, thus allowing efficient transmission of conformational changes to the pore's gate.     

High-Resolution Orientation and Depth of Insertion of the Voltage-Sensing S4 Helix of a Potassium Channel in Lipid Bilayers

The opening and closing of voltage-gated potassium (Kv) channels are controlled by several conserved Arg residues in the S4 helix of the voltage-sensing domain. The interaction of these positively charged Arg residues with the lipid membrane has been of intense interest for understanding how membrane proteins fold to allow charged residues to insert into lipid bilayers against free-energy barriers. Using solid-state NMR, we have now determined the orientation and insertion depth of the S4 peptide of the KvAP channel in lipid bilayers. Two-dimensional ¹⁵N correlation experiments of macroscopically oriented S4 peptide in phospholipid bilayers revealed a tilt angle of 40° and two possible rotation angles differing by 180° around the helix axis. Remarkably, the tilt angle and one of the two rotation angles are identical to those of the S4 helix in the intact voltage-sensing domain, suggesting that interactions between the S4 segment and other helices of the voltage-sensing domain are not essential for the membrane topology of the S4 helix. ¹³C-³¹P distances between the S4 backbone and the lipid ³¹P indicate a ∼ 9 Å local thinning and 2 Å average thinning of the DMPC (1,2-dimyristoyl-sn-glycero-3-phosphochloline)/DMPG (1,2-dimyristoyl-sn-glycero-3-phosphatidylglycerol) bilayer, consistent with neutron diffraction data. Moreover, a short distance of 4.6 Å from the guanidinium C^ζ of the second Arg to ³¹P indicates the existence of guanidinium phosphate hydrogen bonding and salt bridges. These data suggest that the structure of the Kv gating helix is mainly determined by protein-lipid interactions instead of interhelical protein-protein interactions, and the S4 amino acid sequence encodes sufficient information for the membrane topology of this crucial gating helix.     

Modulation of the Voltage-gated Potassium Channel (Kv4.3) and the Auxiliary Protein (KChIP3) Interactions by the Current Activator NS5806

KChIP3 (potassium channel interacting protein 3) is a calcium-binding protein that binds at the N terminus of the Kv4 voltage-gated potassium channel through interactions at two contact sites and has been shown to regulate potassium current gating kinetics as well as channel trafficking in cardiac and neuronal cells. Using fluorescence spectroscopy, isothermal calorimetry, and docking simulations we show that the novel potassium current activator, NS5806, binds at a hydrophobic site on the C terminus of KChIP3 in a calcium-dependent manner, with an equilibrium dissociation constant of 2–5 μm in the calcium-bound form. We further determined that the association between KChIP3 and the hydrophobic N terminus of Kv4.3 is calcium-dependent, with an equilibrium dissociation constant in the apo-state of 70 ± 3 μm and 2.7 ± 0.1 μm in the calcium-bound form. NS5806 increases the affinity between KChIP3 and the N terminus of Kv4.3 (K_d = 1.9 ± 0.1 μm) in the presence and absence of calcium. Mutation of Tyr-174 or Phe-218 on KChIP3 abolished the enhancement of Kv4.3 site 1 binding in the apo-state, highlighting the role of these residues in drug and K4.3 binding. Kinetic studies show that NS5806 decreases the rate of dissociation between KChIP3 and the N terminus of K_V4.3. Overall, these studies support the idea that NS5806 directly interacts with KChIP3 and modulates the interactions between this calcium-binding protein and the T1 domain of the Kv4.3 channels through reorientation of helix 10 on KChIP3.     

Harmonic Analysis of the Fluorescence Response of Bimane Adducts of Colicin E1 at Helices 6, 7, and 10

The pre-channel state of helices 6, 7, and 10 (Val⁴⁴⁷–Gly⁴⁷⁵ and Ile⁵⁰⁸–Ile⁵²²) of colicin E1 was investigated by a site-directed fluorescence labeling technique. A total of 44 cysteine variants were purified and covalently labeled with monobromobimane fluorescent probe. A variety of fluorescence properties of the bimane fluorophore were measured for both the soluble and membrane-bound states of the channel peptide, including the fluorescence emission maximum, fluorescence anisotropy, and membrane bilayer penetration depth. Using site-directed fluorescence labeling combined with our novel helical periodicity analysis method, the data revealed that helices 6, 7, and 10 are separate amphipathic α-helices with a calculated periodicity of T = 3.34 ± 0.08 for helix 6, T = 3.56 ± 0.03 for helix 7, and T = 2.99 ± 0.12 for helix 10 in the soluble state. In the membrane-bound state, the helical periodicity was determined to be T = 3.00 ± 0.15 for helix 6, T = 3.68 ± 0.03 for helix 7, and T = 3.47 ± 0.04 for helix 10. Dual fluorescence quencher analysis showed that both helices 6 and 7 adopt a tilted topology that correlates well with the analysis based on the fluorescence anisotropy profile. These data provide further support for the umbrella model of the colicin E1 channel domain.     

Interaction between Soluble and Membrane-Embedded Potassium Channel Peptides Monitored by Fourier Transform Infrared Spectroscopy

Recent studies have explored the utility of Fourier transform infrared spectroscopy (FTIR) in dynamic monitoring of soluble protein-protein interactions. Here, we investigated the applicability of FTIR to detect interaction between synthetic soluble and phospholipid-embedded peptides corresponding to, respectively, a voltage-gated potassium (Kv) channel inactivation domain (ID) and S4–S6 of the Shaker Kv channel (KV1; including the S4–S5 linker “pre-inactivation” ID binding site). KV1 was predominantly α-helical at 30°C when incorporated into dimyristoyl-l-α-phosphatidylcholine (DMPC) bilayers. Cooling to induce a shift in DMPC from liquid crystalline to gel phase reversibly decreased KV1 helicity, and was previously shown to partially extrude a synthetic S4 peptide. While no interaction was detected in liquid crystalline DMPC, upon cooling to induce the DMPC gel phase a reversible amide I peak (1633 cm⁻¹) consistent with novel hydrogen bond formation was detected. This spectral shift was not observed for KV1 in the absence of ID (or vice versa), nor when the non-inactivating mutant V7E ID was applied to KV1 under similar conditions. Alteration of salt or redox conditions affected KV1-ID hydrogen bonding in a manner suggesting electrostatic KV1-ID interaction favored by a hairpin conformation for the ID and requiring extrusion of one or more KV1 domains from DMPC, consistent with ID binding to S4–S5. These findings support the utility of FTIR in detecting reversible interactions between soluble and membrane-embedded proteins, with lipid state-sensitivity of the conformation of the latter facilitating control of the interaction.     

Positions of the cytoplasmic end of BK α S0 helix relative to S1–S6 and of β1 TM1 and TM2 relative to S0–S6

The large-conductance, voltage- and Ca²⁺-gated K⁺ (BK) channel consists of four α subunits, which form a voltage- and Ca²⁺-gated channel, and up to four modulatory β subunits. The β1 subunit is expressed in smooth muscle, where it slows BK channel kinetics and shifts the conductance–voltage (G-V) curve to the left at [Ca²⁺] > 2 µM. In addition to the six transmembrane (TM) helices, S1–S6, conserved in all voltage-dependent K⁺ channels, BK α has a unique seventh TM helix, S0, which may contribute to the unusual rightward shift in the G-V curve of BK α in the absence of β1 and to a leftward shift in its presence. Such a role is supported by the close proximity of S0 to S3 and S4 in the voltage-sensing domain. Furthermore, on the extracellular side of the membrane, one of the two TM helices of β1, TM2, is adjacent to S0. We have now analyzed induced disulfide bond formation between substituted Cys residues on the cytoplasmic side of the membrane. There, in contrast, S0 is closest to the S2–S3 loop, from which position it is displaced on the addition of β1. The cytoplasmic ends of β1 TM1 and TM2 are adjacent and are located between the S2–S3 loop of one α subunit and S1 of a neighboring α subunit and are not adjacent to S0; i.e., S0 and TM2 have different trajectories through the membrane. In the absence of β1, 70% of disulfide bonding of W43C (S0) and L175C (S2–S3) has no effect on V₅₀ for activation, implying that the cytoplasmic end of S0 and the S2–S3 loop move in concert, if at all, during activation. Otherwise, linking them together in one state would obstruct the transition to the other state, which would certainly change V₅₀.     

Calculation of the Gating Charge for the Kv1.2 Voltage-Activated Potassium Channel

The atomic models of the Kv1.2 potassium channel in the active and resting state, originally presented elsewhere, are here refined using molecular dynamics simulations in an explicit membrane-solvent environment. With a minor adjustment of the orientation of the first arginine along the S4 segment, the total gating charge of the channel determined from >0.5 μs of molecular dynamics simulation is ∼12-12.7 e, in good accord with experimental estimates for the Shaker potassium channel, indicating that the final models offer a realistic depiction of voltage-gating. In the resting state of Kv1.2, the S4 segment in the voltage-sensing domain (VSD) spontaneously converts into a 3₁₀ helix over a stretch of 10 residues. The 3₁₀ helical conformation orients the gating arginines on S4 toward a water-filled crevice within the VSD and allows salt-bridge interactions with negatively charged residues along S2 and S3. Free energy calculations of the fractional transmembrane potential, acting upon key charged residues of the VSD, reveals that the applied field varies rapidly over a narrow region of 10-15 Å corresponding to the outer leaflet of the bilayer. The focused field allows the transfer of a large gating charge without translocation of S4 across the membrane.     

Temperature Dependence of Vasopressin Action on the Toad Bladder

Toad bladders were challenged with vasopressin at one temperature, fixed on the mucosa with 1% glutaraldehyde, and then subjected to an osmotic gradient at another temperature. Thus, the temperature dependence of vasopressin action on membrane permeability was distinguished from the temperature dependence of osmotic water flux. As the temperature was raised from 20° to 38°C, there was a substantial increase in the velocity of vasopressin action, but osmotic flux was hardly affected. In this range of temperature the apparent energy of activation for net water movement across the bladder amounted to only 1.2 kcal/mole, a value well below the activation energy for bulk water viscosity. It is suggested that osmotic water flux takes place through narrow, nonpolar channels in the membrane. When the temperature was raised from 4° to 20°C, both vasopressin action as well as osmotic water flux were markedly enhanced. Activation energies for net water movement were now 8.5 kcal/mole (4°–9°C) and 4.1 kcal/mole (9°–20°C), indicating that the components of the aqueous channel undergo conformational changes as the temperature is lowered from 20°C. At 43°C bladder reactivity to vasopressin was lost, and irreversible changes in selective permeability were observed. The apparent energy of activation for net water movement across the denatured membrane was 6.6 kcal/mole. Approximately 1 µosmol of NaCl was exchanged for 1 µl of H₂O across the denatured membrane.     

Atomic constraints between the voltage sensor and the pore domain in a voltage-gated K+ channel of known structure

In voltage-gated K(+) channels (Kv), membrane depolarization promotes a structural reorganization of each of the four voltage sensor domains surrounding the conducting pore, inducing its opening. Although the crystal structure of Kv1.2 provided the first atomic resolution view of a eukaryotic Kv channel, several components of the voltage sensors remain poorly resolved. In particular, the position and orientation of the charged arginine side chains in the S4 transmembrane segments remain controversial. Here we investigate the proximity of S4 and the pore domain in functional Kv1.2 channels in a native membrane environment using electrophysiological analysis of intersubunit histidine metallic bridges formed between the first arginine of S4 (R294) and residues A351 or D352 of the pore domain. We show that histidine pairs are able to bind Zn(2+) or Cd(2+) with high affinity, demonstrating their close physical proximity. The results of molecular dynamics simulations, consistent with electrophysiological data, indicate that the position of the S4 helix in the functional open-activated state could be shifted by approximately 7-8 A and rotated counterclockwise by 37 degrees along its main axis relative to its position observed in the Kv1.2 x-ray structure. A structural model is provided for this conformation. The results further highlight the dynamic and flexible nature of the voltage sensor.     

The Stoichiometry and Biophysical Properties of the Kv4 Potassium Channel Complex with K+ Channel-interacting Protein (KChIP) Subunits Are Variable,Depending on the Relative Expression Level

Kv4 is a voltage-gated K⁺ channel, which underlies somatodendritic subthreshold A-type current (I_SA) and cardiac transient outward K⁺ (I_to) current. Various ion channel properties of Kv4 are known to be modulated by its auxiliary subunits, such as K⁺ channel-interacting protein (KChIP) or dipeptidyl peptidase-like protein. KChIP is a cytoplasmic protein and increases the current amplitude, decelerates the inactivation, and accelerates the recovery from inactivation of Kv4. Crystal structure analysis demonstrated that Kv4 and KChIP form an octameric complex with four Kv4 subunits and four KChIP subunits. However, it remains unknown whether the Kv4·KChIP complex can have a different stoichiometry other than 4:4. In this study, we expressed Kv4.2 and KChIP4 with various ratios in Xenopus oocytes and observed that the biophysical properties of Kv4.2 gradually changed with the increase in co-expressed KChIP4. The tandem repeat constructs of Kv4.2 and KChIP4 revealed that the 4:4 (Kv4.2/KChIP4) channel shows faster recovery than the 4:2 channel, suggesting that the biophysical properties of Kv4.2 change, depending on the number of bound KChIP4s. Subunit counting by single-molecule imaging revealed that the bound number of KChIP4 in each Kv4.2·KChIP4 complex was dependent on the expression level of KChIP4. Taken together, we conclude that the stoichiometry of Kv4·KChIP complex is variable, and the biophysical properties of Kv4 change depending on the number of bound KChIP subunits.     

Interaction between extracellular Hanatoxin and the resting conformation of the voltage-sensor paddle in Kv channels

In voltage-activated potassium (Kv) channels, basic residues in S4 enable the voltage-sensing domain to move in response to membrane depolarization and thereby trigger the activation gate to open. In the X-ray structure of the KvAP channel, the S4 helix is located near the intracellular boundary of the membrane where it forms a "voltage-sensor paddle" motif with the S3b helix. It has been proposed that the paddle is lipid-exposed and that it translocates through the membrane as it activates. We studied the interaction of externally applied Hanatoxin with the voltage-sensor paddle in Kv channels and show that the toxin binds tightly even at negative voltages where the paddle is resting and the channel is closed. Moreover, measurements of gating charge movement suggest that Hanatoxin interacts with and stabilizes the resting paddle. These findings point to an extracellular location for the resting conformation of the voltage-sensor paddle and constrain its transmembrane movements during activation.     

Dynamics of Kv1 Channel Transport in Axons

Concerted actions of various ion channels that are precisely targeted along axons are crucial for action potential initiation and propagation, and neurotransmitter release. However, the dynamics of channel protein transport in axons remain unknown. Here, using time-lapse imaging, we found fluorescently tagged Kv1.2 voltage-gated K⁺ channels (YFP-Kv1.2) moved bi-directionally in discrete puncta along hippocampal axons. Expressing Kvβ2, a Kv1 accessory subunit, markedly increased the velocity, the travel distance, and the percentage of moving time of these puncta in both anterograde and retrograde directions. Suppressing the Kvβ2-associated protein, plus-end binding protein EB1 or kinesin II/KIF3A, by siRNA, significantly decreased the velocity of YFP-Kv1.2 moving puncta in both directions. Kvβ2 mutants with disrupted either Kv1.2-Kvβ2 binding or Kvβ2-EB1 binding failed to increase the velocity of YFP-Kv1.2 puncta, confirming a central role of Kvβ2. Furthermore, fluorescently tagged Kv1.2 and Kvβ2 co-moved along axons. Surprisingly, when co-moving with Kv1.2 and Kvβ2, EB1 appeared to travel markedly faster than its plus-end tracking. Finally, using fission yeast S. pombe expressing YFP-fusion proteins as reference standards to calibrate our microscope, we estimated the numbers of YFP-Kv1.2 tetramers in axonal puncta. Taken together, our results suggest that proper amounts of Kv1 channels and their associated proteins are required for efficient transport of Kv1 channel proteins along axons.     

Transfer of ion binding site from ether-à-go-go to Shaker: Mg2+ binds to resting state to modulate channel opening

In ether-à-go-go (eag) K⁺ channels, extracellular divalent cations bind to the resting voltage sensor and thereby slow activation. Two eag-specific acidic residues in S2 and S3b coordinate the bound ion. Residues located at analogous positions are ∼4 Å apart in the x-ray structure of a Kv1.2/Kv2.1 chimera crystallized in the absence of a membrane potential. It is unknown whether these residues remain in proximity in Kv1 channels at negative voltages when the voltage sensor domain is in its resting conformation. To address this issue, we mutated Shaker residues I287 and F324, which correspond to the binding site residues in eag, to aspartate and recorded ionic and gating currents in the presence and absence of extracellular Mg²⁺. In I287D+F324D, Mg²⁺ significantly increased the delay before ionic current activation and slowed channel opening with no readily detectable effect on closing. Because the delay before Shaker opening reflects the initial phase of voltage-dependent activation, the results indicate that Mg²⁺ binds to the voltage sensor in the resting conformation. Supporting this conclusion, Mg²⁺ shifted the voltage dependence and slowed the kinetics of gating charge movement. Both the I287D and F324D mutations were required to modulate channel function. In contrast, E283, a highly conserved residue in S2, was not required for Mg²⁺ binding. Ion binding affected activation by shielding the negatively charged side chains of I287D and F324D. These results show that the engineered divalent cation binding site in Shaker strongly resembles the naturally occurring site in eag. Our data provide a novel, short-range structural constraint for the resting conformation of the Shaker voltage sensor and are valuable for evaluating existing models for the resting state and voltage-dependent conformational changes that occur during activation. Comparing our data to the chimera x-ray structure, we conclude that residues in S2 and S3b remain in proximity throughout voltage-dependent activation.     

Surface Expression and Subunit Specific Control of Steady Protein Levels by the Kv7.2 Helix A-B Linker

Kv7.2 and Kv7.3 are the main components of the neuronal voltage-dependent M-current, which is a subthreshold potassium conductance that exerts an important control on neuronal excitability. Despite their predominantly intracellular distribution, these channels must reach the plasma membrane in order to control neuronal activity. Thus, we analyzed the amino acid sequence of Kv7.2 to identify intrinsic signals that may control its surface expression. Removal of the interlinker connecting helix A and helix B of the intracellular C-terminus produces a large increase in the number of functional channels at the plasma membrane. Moreover, elimination of this linker increased the steady-state amount of protein, which was not associated with a decrease of protein degradation. The magnitude of this increase was inversely correlated with the number of helix A – helix B linkers present in the tetrameric channel assemblies. In contrast to the remarkable effect on the amount of Kv7.2 protein, removal of the Kv7.2 linker had no detectable impact on the steady-state levels of Kv7.3 protein.     

Co-assembly of Kv4 α Subunits with K+ Channel-interacting Protein 2 Stabilizes Protein Expression and Promotes Surface Retention of Channel Complexes

Members of the K⁺ channel-interacting protein (KChIP) family bind the distal N termini of members of the Shal subfamily of voltage-gated K⁺ channel (Kv4) pore-forming (α) subunits to generate rapidly activating, rapidly inactivating neuronal A-type (I_A) and cardiac transient outward (I_to) currents. In heterologous cells, KChIP co-expression increases cell surface expression of Kv4 α subunits and Kv4 current densities, findings interpreted to suggest that Kv4·KChIP complex formation enhances forward trafficking of channels (from the endoplasmic reticulum or the Golgi complex) to the surface membrane. The results of experiments here, however, demonstrate that KChIP2 increases cell surface Kv4.2 protein expression (∼40-fold) by an order of magnitude more than the increase in total protein (∼2-fold) or in current densities (∼3-fold), suggesting that mechanisms at the cell surface regulate the functional expression of Kv4.2 channels. Additional experiments demonstrated that KChIP2 decreases the turnover rate of cell surface Kv4.2 protein by inhibiting endocytosis and/or promoting recycling. Unexpectedly, the experiments here also revealed that Kv4.2·KChIP2 complex formation stabilizes not only (total and cell surface) Kv4.2 but also KChIP2 protein expression. This reciprocal protein stabilization and Kv4·KChIP2 complex formation are lost with deletion of the distal (10 amino acids) Kv4.2 N terminus. Taken together, these observations demonstrate that KChIP2 differentially regulates total and cell surface Kv4.2 protein expression and Kv4 current densities.     

Molecular dynamics simulation of Kv channel voltage sensor helix in a lipid membrane with applied electric field

In this article, we present the results of the molecular dynamics simulations of amphiphilic helix peptides of 13 amino-acid residues, placed at the lipid-water interface of dipalmitoylphosphatidylcholine bilayers. The peptides are identical with, or are derivatives of, the N-terminal segment of the S4 helix of voltage-dependent K channel KvAP, containing four voltage-sensing arginine residues (R1-R4). Upon changing the direction of the externally applied electric field, the tilt angle of the wild-type peptide changes relative to the lipid-water interface, with the N-terminus heading up with an outward electric field. These movements were not observed using an octane membrane in place of the dipalmitoylphosphatidylcholine membrane, and were markedly suppressed by 1), substituting Phe located one residue before the first arginine (R1) with a hydrophilic residue (Ser, Thr); or 2), changing the periodicity rule of Rs from at-every-third to at-every-fourth position; or 3), replacing R1 with a lysine residue (K). These and other findings suggest that the voltage-dependent movement requires deep positioning of Rs when the resting (inward) electric field is present. Later, we performed simulations of the voltage sensor domain (S1-S4) of Kv1.2 channel. In simulations with a strong electric field (0.1 V/nm or above) and positional restraints on the S1 and S2 helices, S4 movement was observed consisting of displacement along the S4 helix axis and a screwlike axial rotation. Gating-charge-carrying Rs were observed to make serial interactions with E183 in S1 and E226 in S2, in the outer water crevice. A 30-ns-backward simulation started from the open-state model gave rise to a structure similar to the recent resting-state model, with S4 moving vertically ∼6.7 Å. The energy landscape around the movement of S4 appears very ragged due to salt bridges formed between gating-charge-carrying residues and negatively charged residues of S1, S2, and S3 helices. Overall, features of S3 and S4 movements are consistent with the recent helical-screw model. Both forward and backward simulations show the presence of at least two stable intermediate structures in which R2 and R3 form salt bridges with E183 or E226, respectively. These structures are the candidates for the states postulated in previous gating kinetic models, such as the Zagotta-Hoshi-Aldrich model, to account for more than one transition step per subunit for activation.     

After Embedding in Membranes Antiapoptotic Bcl-XL Protein Binds Both Bcl-2 Homology Region 3 and Helix 1 of Proapoptotic Bax Protein to Inhibit Apoptotic Mitochondrial Permeabilization

Bcl-XL binds to Bax, inhibiting Bax oligomerization required for mitochondrial outer membrane permeabilization (MOMP) during apoptosis. How Bcl-XL binds to Bax in the membrane is not known. Here, we investigated the structural organization of Bcl-XL·Bax complexes formed in the MOM, including the binding interface and membrane topology, using site-specific cross-linking, compartment-specific labeling, and computational modeling. We found that one heterodimer interface is formed by a specific interaction between the Bcl-2 homology 1–3 (BH1–3) groove of Bcl-XL and the BH3 helix of Bax, as defined previously by the crystal structure of a truncated Bcl-XL protein and a Bax BH3 peptide (Protein Data Bank entry 3PL7). We also discovered a novel interface in the heterodimer formed by equivalent interactions between the helix 1 regions of Bcl-XL and Bax when their helical axes are oriented either in parallel or antiparallel. The two interfaces are located on the cytosolic side of the MOM, whereas helix 9 of Bcl-XL is embedded in the membrane together with helices 5, 6, and 9 of Bax. Formation of the helix 1·helix 1 interface partially depends on the formation of the groove·BH3 interface because point mutations in the latter interface and the addition of ABT-737, a groove-binding BH3 mimetic, blocked the formation of both interfaces. The mutations and ABT-737 also prevented Bcl-XL from inhibiting Bax oligomerization and subsequent MOMP, suggesting that the structural organization in which interactions at both interfaces contribute to the overall stability and functionality of the complex represents antiapoptotic Bcl-XL·Bax complexes in the MOM.