Interaction between fast and ultra-slow inactivation in the voltage-gated sodium channel. Does the inactivation gate stabilize the channel structure?

Recently, we reported that mutation A1529D in the domain (D) IV P-loop of the rat skeletal muscle Na(+) channel mu(1) (DIV-A1529D) enhanced entry to an inactivated state from which the channels recovered with an abnormally slow time constant on the order of approximately 100 s. Transition to this "ultra-slow" inactivated state (USI) was substantially reduced by binding to the outer pore of a mutant mu-conotoxin GIIIA. This indicated that USI reflected a structural rearrangement of the outer channel vestibule and that binding to the pore of a peptide could stabilize the pore structure (Hilber, K., Sandtner, W., Kudlacek, O., Glaaser, I. W., Weisz, E., Kyle, J. W., French, R. J., Fozzard, H. A., Dudley, S. C., and Todt, H. (2001) J. Biol. Chem. 276, 27831-27839). Here, we tested the hypothesis that occlusion of the inner vestibule of the Na(+) channel by the fast inactivation gate inhibits ultra-slow inactivation. Stabilization of the fast inactivated state (FI) by coexpression of the rat brain beta(1) subunit in Xenopus oocytes significantly prolonged the time course of entry to the USI. A reduction in USI was also observed when the FI was stabilized in the absence of the beta(1) subunit, suggesting a causal relation between the occurrence of the FI and inhibition of USI. This finding was further confirmed in experiments where the FI was destabilized by introducing the mutations I1303Q/F1304Q/M1305Q. In DIV-A1529D + I1303Q/F1304Q/M1305Q channels, occurrence of USI was enhanced at strongly depolarized potentials and could not be prevented by coexpression of the beta(1) subunit. These results strongly suggest that FI inhibits USI in DIV-A1529D channels. Binding to the inner pore of the fast inactivation gate may stabilize the channel structure and thereby prevent USI. Some of the data have been published previously in abstract form (Hilber, K., Sandtner, W., Kudlacek, O., Singer, E., and Todt, H. (2002) Soc. Neurosci. Abstr. 27, program number 46.12).     

Molecular basis of ion permeability in a voltage‐gated sodium channel

Voltage‐gated sodium channels are essential for electrical signalling across cell membranes. They exhibit strong selectivities for sodium ions over other cations, enabling the finely tuned cascade of events associated with action potentials. This paper describes the ion permeability characteristics and the crystal structure of a prokaryotic sodium channel, showing for the first time the detailed locations of sodium ions in the selectivity filter of a sodium channel. Electrostatic calculations based on the structure are consistent with the relative cation permeability ratios (Na⁺ ≈ Li⁺ ≫ K⁺, Ca²⁺, Mg²⁺) measured for these channels. In an E178D selectivity filter mutant constructed to have altered ion selectivities, the sodium ion binding site nearest the extracellular side is missing. Unlike potassium ions in potassium channels, the sodium ions in these channels appear to be hydrated and are associated with side chains of the selectivity filter residues, rather than polypeptide backbones.     

Role of terminal dipole charges in aggregation of α-helix pair in the voltage gated K+ channel

The voltage sensor domain (VSD) of the potassium ion channel KvAP is comprised of four (S1–S4) α-helix proteins, which are encompassed by several charged residues. Apart from these charges, each peptide α-helix having two inherent equal and opposite terminal dipolar charges behave like a macrodipole. The activity of voltage gated ion channel is electrostatic, where all the charges (charged residues and dipolar terminal charges) interact with each other and with the transmembrane potential. There are evidences that the role of the charged residues dominate the stabilization of the conformation and the gating process of the ion channel, but the role of the terminal dipolar charges are never considered in such analysis. Here, using electrostatic theory, we have studied the role of the dipolar terminal charges in aggregation of the S3b–S4 helix pair of KvAP in the absence of any external field (V = 0). A system attains stability, when its potential energy reaches minimum values. We have shown that the presence of terminal dipole charges (1) change the total potential energy of the charges on S3b–S4, affecting the stabilization of the α-helix pair within the bilayer lipid membrane and (2) the C- and the N-termini of the α-helices favor a different dielectric medium for enhanced stability. Thus, the dipolar terminal charges play a significant role in the aggregation of the two neighboring α-helices.     

NMR assignments of a 48 kDa tetramer of the T1 domain of the mammalian voltage gated potassium channel Kv1.4

The N-terminal cytosolic T1 domain of the mammalian voltage gated potassium channel Kv1.4 is strongly involved in the tetramerization of the Kv1.4 subunit that is required for forming a functional ion channel. The T1 domain forms a stable tetramer of 48 kDa in solution that cannot be dissociated into monomers. In spite of the high molecular mass it was possible to completely assign the backbone and part of the side chain resonances by multidimensional NMR spectroscopy on uniformly ²H, ¹³C, ¹⁵N enriched protein. The secondary structure analysis derived from the chemical shifts is in line with the expectations from X-ray structures of related proteins.     

Study of the residues involved in the binding of β1 to β3 subunits in the sodium channel

The voltage-gated sodium channel (VGSC) is a complex, which is composed of one pore-forming α subunit and at least one β subunit. Up to now, five β subunits are known: β1/β1A, β1B, β2, β3, and β4, encoded by four genes (SCN1B∼SCN4B). It is critical to have a deep understanding of the interaction between β1 and β3 subunits, two subunits which frequently appear in many diseases concurrently. In this study, we had screened out the new template of β1 subunit for homology modelling, which shares higher similarity to β3. Docking studies of the β1 and β3 homology model were conducted, and likely β1 and β3 binding loci were investigated. The results revealed that β1–β3 is more likely to form a di-polymer than β1–β1 based on molecular interaction analysis, including potential energy analysis, Van der Waals (VDW) energy analysis and electrostatic energy analysis, and in addition, consideration of the hydrogen bonds and hydrophobic contacts that are involved. Based on these analyses, the residues His122 and Lys140 of β1 and Glu 66, Asn 131, Asp 118, Glu 120, Glu133, Asn135, Ser 137 of β3 were predicted to play a functional role.     

3₁₀-helix conformation facilitates the transition of a voltage sensor S4 segment toward the down state

The activation of voltage-gated ion channels is controlled by the S4 helix, with arginines every third residue. The x-ray structures are believed to reflect an open-inactivated state, and models propose combinations of translation, rotation, and tilt to reach the resting state. Recently, experiments and simulations have independently observed occurrence of 3₁₀-helix in S4. This suggests S4 might make a transition from α- to 3₁₀-helix in the gating process. Here, we show 3₁₀-helix structure between Q1 and R3 in the S4 segment of a voltage sensor appears to facilitate the early stage of the motion toward a down state. We use multiple microsecond-steered molecular simulations to calculate the work required for translating S4 both as α-helix and transformed to 3₁₀-helix. The barrier appears to be caused by salt-bridge reformation simultaneous to R4 passing the F233 hydrophobic lock, and it is almost a factor-two lower with 3₁₀-helix. The latter facilitates translation because R2/R3 line up to face E183/E226, which reduces the requirement to rotate S4. This is also reflected in a lower root mean-square deviation distortion of the rest of the voltage sensor. This supports the 3₁₀ hypothesis, and could explain some of the differences between the open-inactivated- versus activated-states.     

Identification of an intra-molecular disulfide bond in the sodium channel β1-subunit

The sodium channel β1 subunit is non-covalently associated with the pore-forming α-subunits, and has been proposed to act as a modulator of channel activity, regulator of channel cell surface expression and cell adhesion molecule. Its importance is evident since mutations of the β1 subunit cause neurologic and cardiovascular disorders. The first described β1 subunit mutation is the C121W, that is related to generalized epilepsy with febrile seizures plus (GEFS+), a childhood genetic epilepsy syndrome. This mutation changed a conserved cysteine residue in position 121 into a tryptophan, putatively disrupting a disulfide bridge that should normally maintain the β1 extracellular immunoglobulin-like fold. Using the 2-D-diagonal-SDS-PAGE technique, we demonstrated the existence of this putative disulfide bridge in the Ig-like extracellular domain of the β1 subunit and its disruption in the epileptogenic C121W mutant.     

Electrostatic interaction between inactivation ball and T1–S1 linker region of Kv1.4 channel

Inactivation of potassium channels plays an important role in shaping the electrical signaling properties of nerve and muscle cells. The rapid inactivation of Kv1.4 has been assumed to be controlled by a “ball and chain” inactivation mechanism. Besides hydrophobic interaction between inactivation ball and the channel's inner pore, the electrostatic interaction has also been proved to participate in the “ball and chain” inactivation process of Kv1.4 channel. Based on the crystal structure of Kv1.2 channel, the acidic T1–S1 linker is indicated to be a candidate interacting with the positively charged hydrophilic region of the inactivation domain. In this study, through mutating the charged residues to amino acids of opposite polar, we identified the electrostatic interaction between the inactivation ball and the T1–S1 linker region of Kv1.4 channel. Inserting negatively charged peptide at the amino terminal of Kv1.4 channel further confirmed the electrostatic interaction between the two regions.     

Functional expression of a rat homologue of the voltage gated either á go-go potassium channel reveals differences in selectivity and activation kinetics between the Drosophila channel and its mammalian counterpart.

We have cloned a mammalian (rat) homologue of Drosophila ether á go-go (eag) cDNA, which encodes a distinct type of voltage activated potassium (K) channel. The derived Drosophila and rat eag polypeptides share > 670 amino acids, with a sequence identity of 61%, exhibiting a high degree of similarity at the N-terminus, the hydrophobic core including the pore forming P region and a potential cyclic nucleotide binding site. Rat eag mRNA is specifically expressed in the central nervous system. In the Xenopus oocyte expression system rat eag mRNA gives rise to voltage activated K channels which have distinct properties in comparison with Drosophila eag channels and other voltage activated K channels. Thus, the rat eag channel further extends the known diversity of K channels. Most notably, the kinetics of rat eag channel activation depend strongly on holding membrane potential. Hyperpolarization slows down the kinetics of activation; conversely depolarization accelerates the kinetics of activation. This novel K channel property may have important implications in neural signal transduction allowing neurons to tune their repolarizing properties in response to membrane hyperpolarization.     

An extracellular domain of the accessory β1 subunit is required for modulating BK channel voltage sensor and gate

A family of tissue-specific auxiliary β subunits modulates large conductance voltage- and calcium-activated potassium (BK) channel gating properties to suit their diverse functions. Paradoxically, β subunits both promote BK channel activation through a stabilization of voltage sensor activation and reduce BK channel openings through an increased energetic barrier of the closed-to-open transition. The molecular determinants underlying β subunit function, including the dual gating effects, remain unknown. In this study, we report the first identification of a β1 functional domain consisting of Y74, S104, Y105, and I106 residues located in the extracellular loop of β1. These amino acids reside within two regions of highest conservation among related β1, β2, and β4 subunits. Analysis in the context of the Horrigan-Aldrich gating model revealed that this domain functions to both promote voltage sensor activation and also reduce intrinsic gating. Free energy calculations suggest that the dual effects of the β1 Y74 and S104-I106 domains can be largely accounted for by a relative destabilization of channels in open states that have few voltage sensors activated. These results suggest a unique and novel mechanism for β subunit modulation of voltage-gated potassium channels wherein interactions between extracellular β subunit residues with the external portions of the gate and voltage sensor regulate channel opening.     

Molecular dynamics investigation of the ω-current in the Kv1.2 voltage sensor domains

Voltage sensor domains (VSD) are transmembrane proteins that respond to changes in membrane voltage and modulate the activity of ion channels, enzymes, or in the case of proton channels allow permeation of protons across the cell membrane. VSDs consist of four transmembrane segments, S1-S4, forming an antiparallel helical bundle. The S4 segment contains several positively charged residues, mainly arginines, located at every third position along the helix. In the voltage-gated Shaker K(+) channel, the mutation of the first arginine of S4 to a smaller uncharged amino acid allows permeation of cations through the VSD. These currents, known as ω-currents, pass through the VSD and are distinct from K(+) currents passing through the main ion conduction pore. Here we report molecular dynamics simulations of the ω-current in the resting-state conformation for Kv1.2 and for four of its mutants. The four tested mutants exhibit various degrees of conductivity for K(+) and Cl(-) ions, with a slight selectivity for K(+) over Cl(-). Analysis of the ion permeation pathway, in the case of a highly conductive mutant, reveals a negatively charged constriction region near the center of the membrane that might act as a selectivity filter to prevent permeation of anions through the pore. The residues R1 in S4 and E1 in S2 are located at the narrowest region of the ω-pore for the resting state conformation of the VSD, in agreement with experiments showing that the largest increase in current is produced by the double mutation E1D and R1S.     

Conformational transitions regulate the exposure of a DNA-binding domain in the RuvBL1–RuvBL2 complex

RuvBL1 and RuvBL2, also known as Pontin and Reptin, are AAA+ proteins essential in small nucleolar ribonucloprotein biogenesis, chromatin remodelling, nonsense-mediated messenger RNA decay and telomerase assembly, among other functions. They are homologous to prokaryotic RuvB, forming single- and double-hexameric rings; however, a DNA binding domain II (DII) is inserted within the AAA+ core. Despite their biological significance, questions remain regarding their structure. Here, we report cryo-electron microscopy structures of human double-ring RuvBL1–RuvBL2 complexes at ∼15 Å resolution. Significantly, we resolve two coexisting conformations, compact and stretched, by image classification techniques. Movements in DII domains drive these conformational transitions, extending the complex and regulating the exposure of DNA binding regions. DII domains connect with the AAA+ core and bind nucleic acids, suggesting that these conformational changes could impact the regulation of RuvBL1–RuvBL2 containing complexes. These findings resolve some of the controversies in the structure of RuvBL1–RuvBL2 by revealing a mechanism that extends the complex by adjustments in DII.     

Mechanisms of calpain mediated proteolysis of voltage gated sodium channel α-subunits following in vitro dynamic stretch injury

Although enhanced calpain activity is well documented after traumatic brain injury (TBI), the pathways targeting specific substrate proteolysis are less defined. Our past work demonstrated that calpain cleaves voltage gated sodium channel (NaCh) α-subunits in an in vitro TBI model. In this study, we investigated the pathways leading to NaCh cleavage utilizing our previously characterized in vitro TBI model, and determined the location of calpain activation within neuronal regions following stretch injury to micropatterned cultures. Calpain specific breakdown products of α-spectrin appeared within axonal, dendritic, and somatic regions 6 h after injury, concurrent with the appearance of NaCh α-subunit proteolysis in both whole cell or enriched axonal preparations. Direct pharmacological activation of either NMDA receptors (NMDArs) or NaChs resulted in NaCh proteolysis. Likewise, a chronic (6 h) dual inhibition of NMDArs/NaChs but not L-type voltage gated calcium channels significantly reduced NaCh proteolysis 6 h after mechanical injury. Interestingly, an early, transient (30 min) inhibition of NMDArs alone significantly reduced NaCh proteolysis. Although a chronic inhibition of calpain significantly reduced proteolysis, a transient inhibition of calpain immediately after injury failed to significantly attenuate NaCh proteolysis. These data suggest that both NMDArs and NaChs are key contributors to calpain activation after mechanical injury, and that a larger temporal window of sustained calpain activation needs consideration in developing effective treatments for TBI.     

The sliding-helix voltage sensor: mesoscale views of a robust structure–function relationship

The voltage sensor (VS) domain of voltage-gated ion channels underlies the electrical excitability of living cells. We simulate a mesoscale model of the VS domain to determine the functional consequences of some of its physical elements. Our mesoscale model is based on VS charges, linear dielectrics, and whole-body motion, applied to an S4 "sliding helix." The electrostatics under voltage-clamped boundary conditions are solved consistently using a boundary-element method. Based on electrostatic configurational energy, statistical-mechanical expectations of the experimentally observable relation between displaced charge and membrane voltage are predicted. Consequences of the model are investigated for variations of S4 configuration (α- and 3(10)-helical), countercharge alignment with S4 charges, protein polarizability, geometry of the gating canal, screening of S4 charges by the baths, and fixed charges located at the bath interfaces. The sliding-helix VS domain has an inherent electrostatic stability in the explored parameter space: countercharges present in the region of weak dielectric always retain an equivalent S4 charge in that region but allow sliding movements displacing 3-4 e (0). That movement is sensitive to small energy variations (<2?kT) along the path dependent on a number of electrostatic parameters tested in our simulations. These simulations show how the slope of the relation between displaced charge and voltage could be tuned in a channel.     

Functional expression of the voltage-gated neuronal mammalian potassium channel rat ether à go-go1 in yeast.

It has been shown previously that heterologous expression of inwardly rectifying potassium channels (K+-channels) from plants and mammals in K+-transport defective yeast mutants can restore the ability of growth in media with low [K+]. In this study, the functional expression of an outward rectifying mammalian K+-channel in yeast is presented for the first time. The outward-rectifying mammalian neuronal K+-channel rat ether à go-go channel 1 (rEAG1, Kv 10.1) was expressed in yeast (Saccharomyces cerevisiae) strains lacking the endogenous K+-uptake systems and/or alkali-metal-cation efflux systems. It was found that a truncated channel version, lacking almost the complete intracellular N-terminus (rEAG1 Delta 190) but not the full-length rEAG1, partially complemented the growth defect of K+-uptake mutant cells (trk1,2 Delta tok1 Delta) in media containing low K+ concentrations. The expression of rEAG1 Delta 190 in a strain lacking the cation efflux systems (nha1 Delta ena1-4 Delta) increased the sensitivity to high monovalent cation concentrations. Both phenotypes were observed, when rEAG1 Delta 190 was expressed in a trk1,2 Delta and nha1, ena1-4 Delta mutant strain. In the presence of K+-channel blockers (Cs+, Ba2+ and quinidine), the growth advantage of rEAG1 Delta 190 expressing trk1,2 tok1 Delta cells disappeared, indicating its dependence on functional rEAG1 channels. The results demonstrate that S. cerevisiae is a suitable expression system even for voltage-gated outward-rectifying mammalian K+-channels.     

Interactions between N and C termini of α1C subunit regulate inactivation of CaV1.2 L-type Ca2+ channel

The modulation and regulation of voltage-gated Ca²⁺ channels is affected by the pore-forming segments, the cytosolic parts of the channel, and interacting intracellular proteins. In this study we demonstrate a direct physical interaction between the N terminus (NT) and C terminus (CT) of the main subunit of the L-type Ca²⁺ channel Ca_V1.2, α_1C, and explore the importance of this interaction for the regulation of the channel. We used biochemistry to measure the strength of the interaction and to map the location of the interaction sites, and electrophysiology to investigate the functional impact of the interaction. We show that the full-length NT (amino acids 1-154) and the proximal (close to the plasma membrane) part of the CT, pCT (amino acids 1508-1669) interact with sub-micromolar to low-micromolar affinity. Calmodulin (CaM) is not essential for the binding. The results further suggest that the NT-CT interaction regulates the channel's inactivation, and that Ca²⁺, presumably through binding to calmodulin (CaM), reduces the strength of NT-CT interaction. We propose a molecular mechanism in which NT and CT of the channel serve as levers whose movements regulate inactivation by promoting changes in the transmembrane core of the channel via S1 (NT) or S6 (pCT) segments of domains I and IV, accordingly, and not as a kind of pore blocker. We hypothesize that Ca²⁺-CaM-induced changes in NT-CT interaction may, in part, underlie the acceleration of Ca_V1.2 inactivation induced by Ca²⁺ entry into the cell.     

Plasmin and chymotrypsin have distinct preferences for channel activating cleavage sites in the γ subunit of the human epithelial sodium channel

Proteolytic activation of the epithelial sodium channel (ENaC) involves cleavage of its γ subunit in a critical region targeted by several proteases. Our aim was to identify cleavage sites in this region that are functionally important for activation of human ENaC by plasmin and chymotrypsin. Sequence alignment revealed a putative plasmin cleavage site in human γENaC (K189) that corresponds to a plasmin cleavage site (K194) in mouse γENaC. We mutated this site to alanine (K189A) and expressed human wild-type (wt) αβγENaC and αβγ_K189AENaC in Xenopus laevis oocytes. The γ_K189A mutation reduced but did not abolish activation of ENaC whole cell currents by plasmin. Mutating a putative prostasin site (γ_RKRK178AAAA) had no effect on the stimulatory response to plasmin. In contrast, a double mutation (γ_{RKRK178AAAA;K189A}) prevented the stimulatory effect of plasmin. We conclude that in addition to the preferential plasmin cleavage site K189, the putative prostasin cleavage site RKRK178 may serve as an alternative site for proteolytic channel activation by plasmin. Interestingly, the double mutation delayed but did not abolish ENaC activation by chymotrypsin. The time-dependent appearance of cleavage products at the cell surface nicely correlated with the stimulatory effect of chymotrypsin on ENaC currents in oocytes expressing wt or double mutant ENaC. Delayed proteolytic activation of the double mutant channel with a stepwise recruitment of so-called near-silent channels was confirmed in single-channel recordings from outside-out patches. Mutating two phenylalanines (FF174) in the vicinity of the prostasin cleavage site prevented proteolytic activation by chymotrypsin. This indicates that chymotrypsin preferentially cleaves at FF174. The close proximity of FF174 to the prostasin site may explain why mutating the prostasin site impedes channel activation by chymotrypsin. In conclusion, this study supports the concept that different proteases have distinct preferences for certain cleavage sites in γENaC, which may be relevant for tissue-specific proteolytic ENaC activation.