Shaker Channels Research Articles

Demonstration of ion channel synthesis by isolated squid giant axon provides functional evidence for localized axonal membrane protein translation

Local translation of membrane proteins in neuronal subcellular domains like soma, dendrites and axon termini is well-documented. In this study, we isolated the electrical signaling unit of an axon by dissecting giant axons from mature squids (Dosidicus gigas). Axoplasm extracted from these axons was found to contain ribosomal RNAs, ~8000 messenger RNA species, many encoding the translation machinery, membrane proteins, translocon and signal recognition particle (SRP) subunits, endomembrane-associated proteins, and unprecedented proportions of SRP RNA (~68% identical to human homolog). While these components support endoplasmic reticulum-dependent protein synthesis, functional assessment of a newly synthesized membrane protein in axolemma of an isolated axon is technically challenging. Ion channels are ideal proteins for this purpose because their functional dynamics can be directly evaluated by applying voltage clamp across the axon membrane. We delivered in vitro transcribed RNA encoding native or Drosophila voltage-activated Shaker KV channel into excised squid giant axons. We found that total K+ currents increased in both cases; with added inactivation kinetics on those axons injected with RNA encoding the Shaker channel. These results provide unambiguous evidence that isolated axons can exhibit de novo synthesis, assembly and membrane incorporation of fully functional oligomeric membrane proteins.

Isolation of Aggressive Behavior Mutants in Drosophila Using a Screen for Wing Damage.

Aggression is a complex social behavior that is widespread in nature. To date, only a limited number of genes that affect aggression have been identified, in large part because the complexity of the phenotype makes screening difficult and time-consuming regardless of the species that is studied. We discovered that aggressive group-housed Drosophila melanogaster males inflict damage on each other's wings, and show that wing damage negatively affects their ability to fly and mate. Using this wing-damage phenotype, we screened males from ∼1400 chemically mutagenized strains and found ∼40 mutant strains with substantial wing damage. Five of these mutants also had increased aggressive behavior. To identify the causal mutation in one of our top aggressive strains, we used whole-genome sequencing and genomic duplication rescue strategies. We identified a novel mutation in the voltage-gated potassium channel Shaker (Sh) and show that a nearby previously identified Sh mutation also results in increased aggression. This simple screen can be used to dissect the molecular mechanisms underlying aggression.

Nonreciprocal homeostatic compensation in Drosophila potassium channel mutants.

Homeostatic control of intrinsic excitability is important for long-term regulation of neuronal activity. In conjunction with many other forms of plasticity, intrinsic homeostasis helps neurons maintain stable activity regimes in the face of external input variability and destabilizing genetic mutations. In this study, we report a mechanism by which Drosophila melanogaster larval motor neurons stabilize hyperactivity induced by the loss of the delayed rectifying K+ channel Shaker cognate B (Shab), by upregulating the Ca2+-dependent K+ channel encoded by the slowpoke (slo) gene. We also show that loss of SLO does not trigger a reciprocal compensatory upregulation of SHAB, implying that homeostatic signaling pathways utilize compensatory pathways unique to the channel that was mutated. SLO upregulation due to loss of SHAB involves nuclear Ca2+ signaling and dCREB, suggesting that the slo homeostatic response is transcriptionally mediated. Examination of the changes in gene expression induced by these mutations suggests that there is not a generic transcriptional response to increased excitability in motor neurons, but that homeostatic compensations are influenced by the identity of the lost conductance.NEW & NOTEWORTHY The idea that activity-dependent homeostatic plasticity is driven solely by firing has wide credence. In this report we show that homeostatic compensation after loss of an ion channel conductance is tailored to identity of the channel lost, not its properties.

Analysis of the State-Dependent Block of Shaker IR by bTBuA

Slow inactivation of Shaker-related voltage-gated potassium channels is caused by closure of the inactivation gate in the selectivity filter while the intracellular activation gate (A-gate), that controls ion access to the central pore cavity, is open ([OI] gate configuration). Using internally applied benzyl-tributylammonium (bTBuA) ions and the trap-door paradigm we suggested earlier that the A-gate in Kv4.1 channels might play a novel role as the inactivation gate and being responsible for closed-state inactivation. The current study investigated whether bTBuA shows the characteristics of QA block in slow-inactivated Shaker-IR having open A-gate ([OI]), and therefore, validate the conclusions drawn for Kv4.1. Currents were recorded in excised inside-out patches expressing T449K Shaker-IR channels that displays relatively fast slow inactivation (τi=15 ms). A train of 5.0-s-long depolarizing pulses to +50 mV was applied and the peak currents were determined upon exposure of the intracellular surface of open or open-inactivated channels to 100 µM bTBuA. We found that bTBuA shows a classical open-channel block when applied intracellularly to Shaker channels having both the A-gate and the P-gate open, similarly to when applied to Kv4.1. Next bTBuA was applied to fully inactivated Shaker channels having [OI] configuration (at +50 mV). We found that the compound becomes trapped behind the activation gate upon repolarization: although excess bTBuA was washed out between two pulses, the peak current of the second pulse was greatly reduced indicating trapping of the blocker. On the contrary, bTBuA fails to block inactivated Kv4.1 channels. Our results therefore validate the use of bTBuA as a tool to prove the hypothesis that the A-gate in fully inactivated Kv4.x channels is closed. Futhermore, bTBuA can be locked into Shaker in the absence of the I470C previously shown to be required for trapping of TEA. Support: KTIA_NAP_13-2-2015-0009.

Margatoxin-bound quantum dots as a novel inhibitor of the voltage-gated ion channel Kv1.3.

Venom-derived ion channel inhibitors have strong channel selectivity, potency, and stability; however, tracking delivery to their target can be challenging. Herein, we utilized luminescent quantum dots (QDs) conjugated to margatoxin (MgTx) as a traceable vehicle to target a voltage-dependent potassium channel, Kv1.3, which has a select distribution and well-characterized role in immunity, glucose metabolism, and sensory ability. We screened both unconjugated (MgTx) and conjugated MgTx (QD-MgTx) for their ability to inhibit Shaker channels Kv1.1 to Kv1.7 using patch-clamp electrophysiology in HEK293 cells. Our data indicate that MgTx inhibits 79% of the outward current in Kv1.3-transfected cells and that the QD-MgTx conjugate is able to achieve a similar level of block, albeit a slightly reduced efficacy (66%) and at a slower time course (50% block by 10.9±1.1min, MgTx; vs. 15.3±1.2min, QD-MgTx). Like the unbound peptide, the QD-MgTx conjugate inhibits both Kv1.3 and Kv1.2 at a 1nM concentration, whereas it does not inhibit other screened Shaker channels. We tested the ability of QD-MgTx to inhibit native Kv1.3 expressed in the mouse olfactory bulb (OB). In brain slices of the OB, the conjugate acted similarly to MgTx to inhibit Kv1.3, causing an increased action potential firing frequency attributed to decreased intraburst duration rather than interspike interval. Our data demonstrate a retention of known biophysical properties associated with block of the vestibule of Kv1.3 by QD-MgTx conjugate compared to that of MgTx, inferring QDs could provide a useful tool to deliver ion channel inhibitors to targeted tissues invivo.

K+ channel reorganization and homeostatic plasticity during postembryonic development: biophysical and genetic analyses in acutely dissociated Drosophila central neurons

Intrinsic electric activities of neurons play important roles in establishing and refining neural circuits during development. However, how the underlying ionic currents undergo postembryonic reorganizations remains largely unknown. Using acutely dissociated neurons from larval, pupal, and adult Drosophila brains, we show drastic re-assemblies and compensatory regulations of voltage-gated (IKv) and Ca2+-activated (IK(Ca)) K+ currents during postembryonic development. Larval and adult neurons displayed prominent fast-inactivating IKv, mediated by the Shaker (Sh) channel to a large extent, while in the same neurons IK(Ca) was far smaller in amplitude. In contrast, pupal neurons were characterized by large sustained IKv and prominent IK(Ca), encoded predominantly by the slowpoke (slo) gene. Surprisingly, deletion of Sh in the ShM null mutant removed inactivating, transient IKv from large portions of neurons at all stages. Interestingly, elimination of Sh currents was accompanied by upregulation of non-Sh transient IKv. In comparison, the slo1 mutation abolished the vast majority of IK(Ca), particularly at the pupal stage. Strikingly, the deficiency of IK(Ca) in slo pupae was compensated by the transient component of IKv mediated by Sh channels. Thus, IK(Ca) appears to play critical roles in pupal development and its absence induces functional compensations from a specific transient IKv current. While mutants lacking either Sh or slo currents survived normally, Sh;;slo double mutants deficient in both failed to survive through pupal metamorphosis. Together, our data highlight significant reorganizations and homeostatic compensations of K+ currents during postembryonic development and uncover previously unrecognized roles for Sh and slo in this plastic process.

Efficient enzymatic cyclization of an inhibitory cystine knot-containing peptide.

Disulfide-rich peptides isolated from cone snails are of great interest as drug leads due to their high specificity and potency toward therapeutically relevant ion channels and receptors. They commonly contain the inhibitor cystine knot (ICK) motif comprising three disulfide bonds forming a knotted core. Here we report the successful enzymatic backbone cyclization of an ICK-containing peptide κ-PVIIA, a 27-amino acid conopeptide from Conus purpurascens, using a mutated version of the bacterial transpeptidase, sortase A. Although a slight loss of activity was observed compared to native κ-PVIIA, cyclic κ-PVIIA is a functional peptide that inhibits the Shaker voltage-gated potassium (Kv) channel. Molecular modeling suggests that the decrease in potency may be related to the loss of crucial, but previously unidentified electrostatic interactions between the N-terminus of the peptide and the Shaker channel. This hypothesis was confirmed by testing an N-terminally acetylated κ-PVIIA, which shows a similar decrease in activity. We also investigated the conformational dynamics and hydrogen bond network of cyc-PVIIA, both of which are important factors to be considered for successful cyclization of peptides. We found that cyc-PVIIA has the same conformational dynamics, but different hydrogen bond network compared to those of κ-PVIIA. The ability to efficiently cyclize ICK peptides using sortase A will enable future protein engineering for this class of peptides and may help in the development of novel therapeutic molecules. Biotechnol. Bioeng. 2016;113: 2202-2212. © 2016 Wiley Periodicals, Inc.

The Arabidopsis AtPP2CA Protein Phosphatase Inhibits the GORK K+ Efflux Channel and Exerts a Dominant Suppressive Effect on Phosphomimetic-activating Mutations

The regulation of the GORK (Guard Cell Outward Rectifying) Shaker channel mediating a massive K(+) efflux in Arabidopsis guard cells by the phosphatase AtPP2CA was investigated. Unlike the gork mutant, the atpp2ca mutants displayed a phenotype of reduced transpiration. We found that AtPP2CA interacts physically with GORK and inhibits GORK activity in Xenopus oocytes. Several amino acid substitutions in the AtPP2CA active site, including the dominant interfering G145D mutation, disrupted the GORK-AtPP2CA interaction, meaning that the native conformation of the AtPP2CA active site is required for the GORK-AtPP2CA interaction. Furthermore, two serines in the GORK ankyrin domain that mimic phosphorylation (Ser to Glu) or dephosphorylation (Ser to Ala) were mutated. Mutations mimicking phosphorylation led to a significant increase in GORK activity, whereas mutations mimicking dephosphorylation had no effect on GORK. In Xenopus oocytes, the interaction of AtPP2CA with "phosphorylated" or "dephosphorylated" GORK systematically led to inhibition of the channel to the same baseline level. Single-channel recordings indicated that the GORK S722E mutation increases the open probability of the channel in the absence, but not in the presence, of AtPP2CA. The dephosphorylation-independent inactivation mechanism of GORK by AtPP2CA is discussed in relation with well known conformational changes in animal Shaker-like channels that lead to channel opening and closing. In plants, PP2C activity would control the stomatal aperture by regulating both GORK and SLAC1, the two main channels required for stomatal closure.

The Molecular Mechanism of the Dual Spider Toxin Effect on Voltage Gated K+ Channels

Spider toxins have strong modulatory effects on voltage-gated K+ channels, but their role is strange: depending on the specific channel, toxins can either increase or decrease the current, and it is possible to alter the modulatory effect by transferring the S3b-S4 region between Shaker and Kv2.1 channels. Despite a number of studies of spider toxin effects the exact binding site, orientation, and mechanism of the toxin is unclear, and we have not been able to identify what residues in this region are responsible for the changed modulation.Here, we use electrophysiology and molecular simulations to show that the toxins interact with the S3 segment of the Shaker voltage sensor, but with little or no functional effect in the wild-type Shaker channel. In contrast, the introduction of basic amino acids in the upper region of the S4 segment causes the same toxin to have a strong opening effect on the channel, but neither charges nor other specific mutations explain the difference to Kv2.1. However: by removing only the flexible region of the S3b-S4 loop present in Shaker, the toxin will bind on top of S3b and strongly stabilize the relaxed state of Shaker, which closes the channel with a behavior similar to Kv2.1. This extended loop allows S4 to move relative to S3, which appears to be key for the functional behavior of Shaker, in contrast to Kv2.1 where the activation motion is more concerted. This provides a molecular mechanism for the dual role-of-action of the toxins, and suggests that the length of the S3-S4 loop in different channels could determine whether voltage sensors activate with a sliding S4 segment or S3b-S4 paddle action.

Engineering Subunit and Nonsense Suppression Stoichiometry in Potassium Channels

Multi-subunit membrane proteins employ chemically diverse inter- and intramolecular contacts to regulate their function. Potassium channel slow inactivation relies on a hydrogen bond network around the selectivity filter which supports an internal timing mechanism to control the transition between conducting and non-conducting conformations. Previously we used targeted replacement of W434 with the isosteric non-canonical amino acid 2-amino-3-indol-1-yl-propionic acid (Ind) to highlight the importance of the indole H-bond of this highly conserved Trp residue. However, these experiments were unable to nuance the specific contributions of individual channel subunits to channel inactivation. We therefore designed an engineered protein approach whereby complementary intein sequences are incorporated into the N- or C- termini of Shaker monomers, respectively, flanked by previously identified endoplasmic reticulum (ER) retention signals. The data shows that functional channels composed of monomeric ER-intein subunits are restricted from the surface membrane. However, co-expression of complementary ER-intein monomers results in robust expression of functional K+ channels through intein ligation and excision of both the ER retention signals and intein sequences. This technique was used to express Shaker channels with two W434Ind monomers within a single tetramer, producing an intermediate inactivation rate, consistent with the breakage of multiple H-bonds required for full inactivation. In addition, the fidelity of the stoichiometric assembly was assayed by TEA block of channels with zero, two or four T449F mutations. The development of this approach allows for regulation of subunit stoichiometry in multimeric complexes and advances the use of nonsense suppression by demonstrating a solution to incorporate multiple non-canonical amino acids into a single peptide and multimeric complexes. Specifically, the reduction of overall protein expression that is normally compounded at the second suppression site is effectively abrogated because two transcripts are suppressed independently then ligated as proteins.

A single-point mutation enhances dual functionality of a scorpion toxin

Scorpion venom represents a tremendous, hitherto partially explored peptide library that has been proven to be useful not only for understanding ion channels but also for drug design. MeuTXKα3 is a functionally unknown scorpion toxin-like peptide. Here we describe new transcripts of this gene arising from alternative polyadenylation and its biological function as well as a mutant with a single-point substitution at site 30. Native-like MeuTXKα3 and its mutant were produced in Escherichia coli and their toxic function against Drosophila Shaker K+ channel and its mammalian counterparts (rKv1.1–rKv1.3) were assayed by two-electrode voltage clamp technique. The results show that MeuTXKα3 is a weak toxin with a wide-spectrum of activity on both Drosophila and mammalian K+ channels. The substitution of a proline at site 30 by an asparagine, an evolutionarily conserved functional residue in the scorpion α-KTx family, led to an increased activity on rKv1.2 and rKv1.3 but a decreased activity on the Shaker channel without changing the potency on rKv1.1, suggesting a key role of this site in species selectivity of scorpion toxins. MeuTXKα3 was also active on a variety of bacteria with lethal concentrations ranging from 4.66 to 52.01μM and the mutant even had stronger activity on some of these bacterial species. To the best of our knowledge, this is the first report on a bi-functional short-chain peptide in the lesser Asian scorpion venom. Further extensive mutations of MeuTXKα3 at site 30 could help improve its K+ channel-blocking and antibacterial functions.

Pore dimensions and the role of occupancy in unitary conductance of Shaker K channels.

K channels mediate the selective passage of K(+) across the plasma membrane by means of intimate interactions with ions at the pore selectivity filter located near the external face. Despite high conservation of the selectivity filter, the K(+) transport properties of different K channels vary widely, with the unitary conductance spanning a range of over two orders of magnitude. Mutation of Pro475, a residue located at the cytoplasmic entrance of the pore of the small-intermediate conductance K channel Shaker (Pro475Asp (P475D) or Pro475Gln (P475Q)), increases Shaker's reported ∼ 20-pS conductance by approximately six- and approximately threefold, respectively, without any detectable effect on its selectivity. These findings suggest that the structural determinants underlying the diversity of K channel conductance are distinct from the selectivity filter, making P475D and P475Q excellent probes to identify key determinants of the K channel unitary conductance. By measuring diffusion-limited unitary outward currents after unilateral addition of 2 M sucrose to the internal solution to increase its viscosity, we estimated a pore internal radius of capture of ∼ 0.82 Å for all three Shaker variants (wild type, P475D, and P475Q). This estimate is consistent with the internal entrance of the Kv1.2/2.1 structure if the effective radius of hydrated K(+) is set to ∼ 4 Å. Unilateral exposure to sucrose allowed us to estimate the internal and external access resistances together with that of the inner pore. We determined that Shaker resistance resides mainly in the inner cavity, whereas only ∼ 8% resides in the selectivity filter. To reduce the inner resistance, we introduced additional aspartate residues into the internal vestibule to favor ion occupancy. No aspartate addition raised the maximum unitary conductance, measured at saturating [K(+)], beyond that of P475D, suggesting an ∼ 200-pS conductance ceiling for Shaker. This value is approximately one third of the maximum conductance of the large conductance K (BK) channel (the K channel of highest conductance), reducing the energy gap between their K(+) transport rates to ∼ 1 kT. Thus, although Shaker's pore sustains ion translocation as the BK channel's does, higher energetic costs of ion stabilization or higher friction with the ion's rigid hydration cage in its narrower aqueous cavity may entail higher resistance.

Alternative splicing modulates Kv channel clustering through a molecular ball and chain mechanism.

Ion channel clustering at the post-synaptic density serves a fundamental role in action potential generation and transmission. Here, we show that interaction between the Shaker Kv channel and the PSD-95 scaffold protein underlying channel clustering is modulated by the length of the intrinsically disordered C terminal channel tail. We further show that this tail functions as an entropic clock that times PSD-95 binding. We thus propose a 'ball and chain' mechanism to explain Kv channel binding to scaffold proteins, analogous to the mechanism describing channel fast inactivation. The physiological relevance of this mechanism is demonstrated in that alternative splicing of the Shaker channel gene to produce variants of distinct tail lengths resulted in differential channel cell surface expression levels and clustering metrics that correlate with differences in affinity of the variants for PSD-95. We suggest that modulating channel clustering by specific spatial-temporal spliced variant targeting serves a fundamental role in nervous system development and tuning.

The Role of the Ion Dehydration Process in Low and High Conductance K Channels

Potassium channels are known for exhibiting a wide conductance rate from 8.2 pS to 250 pS despite that selectivity filter is largely conserved. Some aspects about the relation between the charge distribution along to the pore and the conductance rate have been studied. In Shaker channel, a low conductance K-channel, one single point mutation of the P475D increases the 4-fold the maximal unitary conductance. At the same time, BK channels, a large conductance K-channel, has been demonstrated that a ring of negative charged residues in the intracellular entrance are key residues in the control of the maximal ion transport rate. Another main difference between low and high conductance K-channels is the size of the channel pore, BK has a much wider internal vestibule than Shaker. In order to understand at the molecular level the conduction process in both K+ channel classes, we performed molecular dynamics simulations using an applied external electric field to compare the structural properties of BK and Shaker channels (+300 mV, +600 mV and +800mV). Different ranges of currents were observed in Shaker and BK under the influence of the external electric field. At all the voltages tested, BK present the largest rate of ion translocation. Interestingly, K+ ions moving from the intracellular side up to the S4 site show a dehydration pattern, which is more efficient in BK channels. Other channel structural properties are discussed in the context of the dehydration process of the K ions in Shaker and BK.Acknowledgements: CINV is a Millennium Institute. R.S thanks CONICYT doctoral fellowship and project FONDECYT 1110430 (RL), 1131003 (FGN), 1120819 (DN) and proyecto Anillo ACT1107 (FGN)

External Protons Destabilize the Relaxed State of the hERG Channel Voltage Sensor

Voltage sensor relaxation may contribute to the slow deactivation kinetics of hERG channels1. Relaxation stabilizes the voltage sensor in the activated state resulting in a mode-shift, where the voltage-dependence of channel deactivation is shifted ∼-30 mV compared with that of channel activation. Voltage clamp fluorimetry (VCF) reports of voltage sensor movement show a similar shift in the voltage-dependence of S4 return compared with its activation. However, the mechanism underlying voltage sensor relaxation in hERG is unknown. In Shaker channels, voltage sensor relaxation has been shown to be sensitive to the length and composition of the S3-S4 linker2. Here, we assessed the role of S3-S4 linker length and composition in voltage sensor relaxation in hERG channels using two constructs: a chimera with the 9 residue hERG S3-S4 linker substituted with the 31 residue Shaker S3-S4 linker; the other with the 9 residues of the hERG S3-S4 linker mutated to glycine residues. In both cases, the robust shifts in the voltage-dependence of ionic current deactivation relative to that of activation were preserved (−35 and −29 mV in the 31 residue Shaker linker construct (n=4) and the 9 glycine construct (n=5), respectively), suggesting that length and composition of the S3-S4 linker are not important mediators of hERG channel relaxation. Instead, we demonstrate that the ionic current mode-shift is reduced by external protons in a pH-dependent manner with a pKa of 5.5 (n=5). Furthermore, VCF recordings of voltage sensor movement show that acidic pH directly inhibits voltage sensor relaxation. These data suggest that external protons modify voltage sensor relaxation by destabilizing the relaxed conformation and reveal a mechanism by which acidic pH may accelerate hERG channel deactivation gating kinetics. 1.Tan et al., J.Gen.Physiol. 140:293-306, 2012 2.Priest et al., Biophys.J. 105:2312-2322, 2013

Evolutionary Origins of the Shaker Family of Voltage-Gated Potassium Channels

The Shaker family of voltage-gated K+ channels in bilaterians consists of four closely related gene subfamilies (Shaker, Shab, Shaw and Shal) that regulate neuronal excitability in diverse ways. We examined the evolutionary origins of the gene family by characterizing Shaker channels in early branching metazoan lineages. Shaker family channels can be definitively identified by the combination of a voltage-dependent potassium channel core and a cytosolic domain (T1) that mediates subfamily-specific tetrameric assembly. The Shaker, Shab, Shaw and Shal subfamilies are present in cnidarians, a close sister group to the bilaterians. We show that all four subfamilies are highly conserved on a functional level in the sea anemone Nematostella vectensis, an emerging cnidarian model organism. The Nematostella Shaker, Shal and Shaw subfamilies include cnidarian-specific expansions of regulatory subunits that extend functional diversity through the formation of heteromeric channels. We were unable to identify Shaker family genes in choanoflagellates or sponges, but found over 40 Shaker family genes in a ctenophore (Mnemiopsis leidyi). Ctenophores are the earliest branching metazoans with a nervous system. Phylogenetic analysis indicates that the ctenophore Shaker family expanded independently and diverged prior to emergence of the Shaker, Shab, Shal and Shaw subfamilies. The phylogeny and functional analysis support Shaker as the oldest of these subfamilies. Two Mnemiopsis Shaker family subunits heteromerize with Nematostella and mouse Shaker subfamily channels, but not with Shab, Shaw or Shal. Thus the subunit interface of Shaker subfamily may retain ancestral features. Our results indicate that the Shaker family is metazoan-specific and was present by the time neurons evolved. However, the Shaker, Shab, Shal and Shaw subfamilies evolved in the cnidarian/bilaterian lineage long after the evolution of the first nervous systems.

High Temperature Sensitivity is Intrinsic to Voltage-Gated Potassium Channels

Temperature-sensitive transient receptor potential (TRP) ion channels are members of the large tetrameric cation channels superfamily but are considered to be uniquely sensitive to heat, which has been presumed to be due to the existence of an unidentified temperature-sensing domain. We find that the homologous voltage-gated potassium (Kv) channels also exhibit high temperature sensitivity comparable to that of TRPV1, which is detectable under specific conditions when the voltage sensor is functionally decoupled from the activation gate through either intrinsic mechanisms or mutations. Interestingly, mutations could tune Shaker channel to be either heat-activated or heat-deactivated. Therefore, high temperature sensitivity is intrinsic to both TRP and Kv channels. Our findings suggest important physiological roles of heat-induced variation in Kv channel activities. Mechanistically our findings indicate that temperature-sensing TRP channels may not contain a specialized heat-sensor domain; instead, non-obligatory allosteric gating permits the intrinsic heat sensitivity to drive channel activation, allowing temperature-sensitive TRP channels to function as polymodal nociceptors.

Role of the Voltage Sensing Domain S1-S4 in TRPV1 Channels

Transient Receptor Potential Vallinoid type 1 (TRPV1) channel is a voltage-, heat-, and ligand-activated, nonselective cation channel. As a member of hexahelical cation channel superfamily, TRPV1 does not feature the positively charged S4, which is responsible for the voltage sensitivity of voltage-gated ion channels (VGICs). The origin for TRPV1's voltage sensitivity must lie elsewhere. The objective of this study was to investigate the role of “classical” S1-S4 voltage sensor, in particular S4, in the TRPV1, and obtain molecular information about the functional coupling between voltage, chemical and temperature sensors. To test whether the positive charge is involved in activation and to explore the function of the S4 segment in TRPV1 activation, we made a series of single point arginine replacements that scanned residues from E536 through T556 in the S4 and characterized their response to capsaicin, protons and heat using cut-open oocyte voltage-clamp. We also constructed TRPV1/Shaker chimeras in which the S1-S4, S3-S4, or S4 segments of the TRPV1 channel were replaced by the corresponding segments of Shaker channels. The introduction of positively charged residues in the S4 segment resulted in a steeper activation curve and a defective response to capsaicin and protons. Importantly, several of the mutant channels displayed strong inhibition of hyperpolarization-activated inward currents. Three chimeras gave rise to functional channels that exhibited stronger voltage dependence at positive voltages than TRPV1. The chimeras progressively lost inward current with increasing portions replaced by the corresponding Shaker region. They were activated by protons with higher sensitivity compared with TRPV1. These results suggest that re-established outward rectification was dominant over other gating stimuli; in other words, keeping the S1-S4 in the “deactivated” state prevented activation by protons, capsaicin or temperature. In wildtype TRPV1, the S4 seems to inhibit activation by protons.

Capturing distinct KCNQ2 channel resting states by metal ion bridges in the voltage-sensor domain.

Although crystal structures of various voltage-gated K(+) (Kv) and Na(+) channels have provided substantial information on the activated conformation of the voltage-sensing domain (VSD), the topology of the VSD in its resting conformation remains highly debated. Numerous studies have investigated the VSD resting state in the Kv Shaker channel; however, few studies have explored this issue in other Kv channels. Here, we investigated the VSD resting state of KCNQ2, a K(+) channel subunit belonging to the KCNQ (Kv7) subfamily of Kv channels. KCNQ2 can coassemble with the KCNQ3 subunit to mediate the IM current that regulates neuronal excitability. In humans, mutations in KCNQ2 are associated with benign neonatal forms of epilepsy or with severe epileptic encephalopathy. We introduced cysteine mutations into the S4 transmembrane segment of the KCNQ2 VSD and determined that external application of Cd(2+) profoundly reduced the current amplitude of S4 cysteine mutants S195C, R198C, and R201C. Based on reactivity with the externally accessible endogenous cysteine C106 in S1, we infer that each of the above S4 cysteine mutants forms Cd(2+) bridges to stabilize a channel closed state. Disulfide bonds and metal bridges constrain the S4 residues S195, R198, and R201 near C106 in S1 in the resting state, and experiments using concatenated tetrameric constructs indicate that this occurs within the same VSD. KCNQ2 structural models suggest that three distinct resting channel states have been captured by the formation of different S4-S1 Cd(2+) bridges. Collectively, this work reveals that residue C106 in S1 can be very close to several N-terminal S4 residues for stabilizing different KCNQ2 resting conformations.

Archaerhodopsin voltage imaging: synaptic calcium and BK channels stabilize action potential repolarization at the Drosophila neuromuscular junction.

The strength and dynamics of synaptic transmission are determined, in part, by the presynaptic action potential (AP) waveform at the nerve terminal. The ion channels that shape the synaptic AP waveform remain essentially unknown for all but a few large synapses amenable to electrophysiological interrogation. The Drosophila neuromuscular junction (NMJ) is a powerful system for studying synaptic biology, but it is not amenable to presynaptic electrophysiology. Here, we demonstrate that Archaerhodopsin can be used to quantitatively image AP waveforms at the Drosophila NMJ without disrupting baseline synaptic transmission or neuromuscular development. It is established that Shaker mutations cause a dramatic increase in neurotransmitter release, suggesting that Shaker is predominantly responsible for AP repolarization. Here we demonstrate that this effect is caused by a concomitant loss of both Shaker and slowpoke (slo) channel activity because of the low extracellular calcium concentrations (0.2-0.5 mM) used typically to assess synaptic transmission in Shaker. In contrast, at physiological extracellular calcium (1.5 mM), the role of Shaker during AP repolarization is limited. We then provide evidence that calcium influx through synaptic CaV2.1 channels and subsequent recruitment of Slo channel activity is important, in concert with Shaker, to ensure proper AP repolarization. Finally, we show that Slo assumes a dominant repolarizing role during repetitive nerve stimulation. During repetitive stimulation, Slo effectively compensates for Shaker channel inactivation, stabilizing AP repolarization and limiting neurotransmitter release. Thus, we have defined an essential role for Slo channels during synaptic AP repolarization and have revised our understanding of Shaker channels at this model synapse.