Xenopus Leavis Oocytes Research Articles

Observing rapid phosphatidylinositol 3,4-bisphosphate kinetics during activation of voltage-sensing phosphatase (VSP) in mammalian cells using total internal reflection microscopy at high frame rates.

Voltage-sensing phosphatases (VSPs) are PI(4,5)P2/PI(3,4,5)P3-5- and PI(3,4)P2/PI(3,4,5)P3-3-phosphatases consisting of a voltage sensor domain and a catalytic domain (Halaszovich 2009; Kurokawa 2012). Their catalytic activity is controlled by membrane voltage. The effect of VSPs on PI(3,4)P2 is paradox, as it is produced by the 5-phosphatase activity as well as degraded by the 3-phosphatase activity. Grimm & Isacoff (2016) report a transient increase in PI(3,4)P2 immediately after VSP activation in Ciona intestinalis (Ci-) VSP expressing Xenopus leavis oocytes, detectable using a membrane anchored FRET-based PI(3,4)P2 probe, whereas a diffusible, translocating fluorescent probe could not detect the transient.

The Mechanism of NMDA Receptor Hyperexcitation in High Pressure Helium and Hyperbaric Oxygen.

Professional divers exposed to pressures greater than 1.1 MPa may suffer from the high pressure neurological syndrome (HPNS). Divers who use closed-circuit breathing apparatus face the risk of CNS hyperbaric oxygen toxicity (HBOTox). Both syndromes are characterized by reversible CNS hyperexcitability, accompanied by cognitive and motor deficits. Previous studies have demonstrated that the hyperexcitability of HPNS is induced mainly by NMDA receptors (NMDARs). In our recent studies, we demonstrated that the response of NMDARs containing GluN1 + GluN2A subunits was increased by up to 50% at high pressure (HP) He, whereas GluN1 + GluN2B NMDARs response was not affected under similar conditions. Our aim was to compare the responses of both types of NMDARs under HBOTox conditions to those of HP He and to reveal their possible underlying molecular mechanism(s). The two combinations of NMDARs were expressed in Xenopus laevis oocytes, placed in a pressure chamber, voltage-clamped, and their currents were tested at 0.1 (control) −0.54 MPa 100% O2 or 0.1–5.1 MPa He pressures. We show, for the first time, that NMDARs containing the GluN2A subunit exhibit increased responses in 100% O2 at a pressure of 0.54 MPa, similar to those observed in 5.1 MPa He. In contrast, the GluN1 + GluN2B response is not sensitive to either condition. We discovered that neither condition produced statistically significant changes in the voltage-dependent Mg2+ inhibition of the response. The averaged IC50 remained the same, but a higher [Mg2+]o was required to restore the current to its control value. The application of TPEN, a Zn2+ chelator, in control, HP He and HBOTox conditions, revealed that the increase in GluN1 + GluN2A current is associated with the removal of the high-affinity voltage-independent Zn2+ inhibition of the receptor. We propose that HPNS and HBOTox may share a common mechanism, namely removal of Zn2+ from its specific binding site on the N-terminal domain of the GluN2A subunit, which increases the pore input-conductance and produces larger currents and consequently a hyperexcitation.

NaStEP, an essential protein for self-incompatibility in Nicotiana, performs a dual activity as a proteinase inhibitor and as a voltage-dependent channel blocker

The S-specific pollen rejection response in Nicotiana depends on the interaction between S-RNase and a suite of SLF proteins. However, the biochemical pathway requires other essential proteins. One of them is the stigmatic protein NaStEP, which belongs to the Kunitz-type protease inhibitor family. Within the pollen tubes, NaStEP is a positive regulator of HT-B stability, likely inhibiting its degradation and, additionally, interacts with NaSIPP, a mitochondrial phosphate carrier. To gain a deeper understanding of the biochemical role of NaStEP in pollen rejection, we evaluated whether the activity of NaStEP as protease inhibitor is specific to a particular type of protease and whether it has the function of a voltage-dependent channel (VDC) blocker. Our findings indicate that, in vitro, NaStEP inhibits a subtilisin-like protease in an irreversible manner, but not other proteases, such as thermolysin and papain. Furthermore, we found that subtilisin processes the native NaStEP (24 kDa) into two lower molecular weight peptides of 21 and 14 kDa. Moreover, when we incubated NaStEP along with Xenopus leavis oocytes expressing the voltage-dependent potassium channel Kv 1.3, the current was blocked, indicating that NaStEP acts as a VDC blocker. These data allow us to propose NaStEP acts as a key molecule with two functions, one protecting HT-B from degradation by inhibiting a subtilisin-like protease and the second one by forming a complex with a mitochondrial VDC that could destabilize the mitochondria to trigger cell death, which would reinforce S-specific pollen rejection in Nicotiana.

Identification of a Modulatory Site of Action for the Volatile Anesthetic Isoflurane in TREK1 Tandem Pore Potassium Channels

Many members of the K2P family of potassium channels are modulated by volatile anesthetic (VA) agents. While individual residues within a subset of K2P channels are known to be important for anesthetic modulation, those actually contributing to the VA binding site have not yet been revealed. Using a validated photoaffinity analog of the haloether VA isoflurane (azi-isoflurane), we identified a VA binding site within the TREK1 K2P channel. Azi-isoflurane was reacted with recombinantly expressed, purified and reconstituted TREK1 protein, and modified residues were detected by liquid chromatography-tandem mass spectrometry. Two TREK1 residues are modified by Azi-isoflurane: a glycine in the inner helix bundle crossing region of TM2 and a membrane exposed residue in the TM2/TM3 loop. Protection from azi-isoflurane modification by photolabeling in the presence of 100-fold excess isoflurane was only observed for the inner helix bundle glycine, suggesting that the parent VA isoflurane specifically binds in this region. Two electrode voltage clamp studies on TREK1-expressing Xenopus leavis oocytes demonstrate that mutagenesis of the identified inner helix glycine to tryptophan causes a marked increase in channel activity and eliminates TREK1 anesthetic sensitivity. In molecular dynamics simulations, an azi-isoflurane molecule placed in the TREK1 inner helix bundle region makes stable contacts with this glycine as well as a TM3 residue known to modulate anesthetic responsiveness of TASK K2P channels. Mutating the glycine to tryptophan substantially decreases azi-isoflurane binding affinity, as calculated using free energy perturbation. Future work combining photoaffinity labeling, functional characterization, and MD simulation will aim to understand the mechanism by which anesthetic binding at this region leads to channel activation, whether all VAs share this modulatory site, and whether the identified modulatory site is shared by all anesthetic sensitive K2P channels.

Boron Uptake Assay in Xenopus laevis Oocytes.

Boron (B) is essential for plant growth and taken up by plant roots as boric acid. Under B limitation, B uptake and translocation in plants are dependent on the boric acid channels located in the plasma membrane. Xenopus leavis oocyte is a reliable heterologous expression system to characterize transport activities of boric acid channels and related major intrinsic proteins (aquaporins). Here, we outline the protocols for expression of boric acid channels and boric acid uptake assay in Xenopus leavis oocytes.

Two P2X1 receptor transcripts able to form functional channels are present in most human monocytes

To characterize the presence and general properties of P2X1 receptors in single human monocytes we used RT-PCR, flow cytometry, and the patch-clamp and the two-electrode voltage-clamp techniques. Most human monocytes expressed the canonical P2X1 (90%) and its splicing variant P2X1del (88%) mRNAs. P2X1 receptor immunoreactivity was also observed in 70% of these cells. Currents mediated by P2X1 (EC50=1.9±0.8µm) and P2X1del (EC50 >1000µm) channels, expressed in Xenopus leavis oocytes, have different ATP sensitivity and kinetics. Both currents mediated by P2X1 and P2X1del channels kept increasing during the continuous presence of high ATP concentrations. Currents mediated by the native P2X1 receptors in human monocytes showed an EC50=6.3±0.2µm. Currents have kinetics that resemble those observed for P2X1 and P2X1del receptors in oocytes. Our study is the first to demonstrate the expression of P2X1 transcript and its splicing variant P2X1del in most human monocytes. We also, for the first time, described functional homomeric P2X1del channels and demonstrated that currents mediated by P2X1 or P2X1del receptors, during heterologous expression, increased in amplitude when activated with high ATP concentrations in a similar fashion to those channels that increase their conductance under similar conditions, such as P2X7, P2X2, and P2X4 channels.

The Diversity of Light-Driven Ion Pumps and their Conversion into Ion Channels

Microbial rhodopsins are integral seven-transmembrane helix proteins, which covalently bind all-trans-retinal as light-sensitive chromophore. They play an extremely important role in neuroscience as optogenetic tools for the modulation of neuronal activity. These rhodopsins are subdivided into sensory rhodopsins, ion channels and ion pumps.Light-driven ion pumps transport protons, sodium or chloride across the plasma membrane against an electrochemical gradient. We analyzed a variety of ion pumps using two-microelectrode voltage-clamp measurements (TEVC) in Xenopus leavis oocytes. We found that all pumps show different electrophysiological behaviors. Especially light-driven proton pumps can be subdivided according to their characteristics at high electrochemical load, i.e. low extracellular pH and negative voltage. Photocurrents of Bacteriorhodopsin and the rhodopsin from the eukaryotic microalga Coccomyxa subellipsoidea (CsR) are always outward directed or inactivate at high load. In contrast, the rhodopsins from Exiguobacterium sibiricum (ESR) and from the cyanobacterium Gloeobacter violaceus (GR) show inward directed photocurrents at high load. The well-established rhodopsin Arch3 from the archaeon Halorubrum sodomense shows weak inward directed photocurrents at high load.Most recently, the class of sodium pumps was discovered, but so far their underlying mechanism and ion selectivity is poorly understood. Here, we show the first more comprehensive electrophysiological characterization of the light-driven sodium-pumps in native membranes. They are characterized by an unexpected strong pH-dependency. In earlier work we have shown that proton pumps can be converted into operational proton channels via the introduction of selected mutations. Now, we transferred this approach to the sodium pumps and attained also inward directed photocurrents. This observation opens the opportunity to engineer new optogenetic tools with high ion-selectivity.

Differential effects of the recombinant toxin PnTx4(5-5) from the spider Phoneutria nigriventer on mammalian and insect sodium channels.

The toxin PnTx4(5-5) from the spider Phoneutria nigriventer is extremely toxic/lethal to insects but has no macroscopic behavioral effects observed in mice after intracerebral injection. Nevertheless, it was demonstrated that it inhibits the N-methyl-d-aspartate (NMDA) - subtype of glutamate receptors of cultured rat hippocampal neurons. PnTx4(5-5) has 63% identity to PnTx4(6-1), another insecticidal toxin from P.nigriventer, which can slow down the sodium current inactivation in insect central nervous system, but has no effect on Nav1.2 and Nav1.4 rat sodium channels. Here, we have cloned and heterologous expressed the toxin PnTx4(5-5) in Escherichia coli. The recombinant toxin rPnTx4(5-5) was tested on the sodium channel NavBg from the cockroach Blatella germanica and on mammalian sodium channels Nav1.2-1.6, all expressed in Xenopus leavis oocytes. We showed that the toxin has different affinity and mode of action on insect and mammalian sodium channels. The most remarkable effect was on NavBg, where rPnTx4(5-5) strongly slowed down channel inactivation (EC50=212.5nM), and at 1μM caused an increase on current peak amplitude of 105.2±3.1%. Interestingly, the toxin also inhibited sodium current on all the mammalian channels tested, with the higher current inhibition on Nav1.3 (38.43±8.04%, IC50=1.5μM). Analysis of activation curves on Nav1.3 and Nav1.5 showed that the toxin shifts channel activation to more depolarized potentials, which can explain the sodium current inhibition. Furthermore, the toxin also slightly slowed down sodium inactivation on Nav1.3 and Nav1.6 channels. As far as we know, this is the first araneomorph toxin described which can shift the sodium channel activation to more depolarized potentials and also slows down channel inactivation.

Limitation of Light Driven Proton Pumps at High Electrochemical Load

All known light-driven ion pumps are microbial type rhodopsins that transport protons, sodium or chloride across the plasma membrane. Bacteriorhodopsin (BR) from Halobacterium salinarum is the most widely studied representative that has been investigated mainly by spectroscopic and structural methods. Recently light-driven pumps have been employed in optogenetics for silencing of neuronal activity and for voltage sensing (Arch3) and thus received new attention. Surprisingly, the knowledge about the electrophysiological properties of ion pumps in living cells is still poor. The voltage dependence and the limitation of the pumping activity at high electrochemical load, i.e. negative voltage and low pH, are unknown for most light-driven transporters. We will present experiments on rhodopsins from the alga Chlorella vulgaris (CvRh) and the proteorhodopsin-like pump from cyanobacterium Gloeobacter violaceus (GR). Both exhibit in comparison to BR much larger photocurrents in two-microelectrode voltage-clamp measurements (TEVC) on Xenopus leavis oocytes. We identified the reprotonating E132 in GR (D96 in BR) as the position that mediates high photocycle kinetics but is also responsible for the leakiness of proteorhodopsins at high load. Moreover, the proton shuttle R83 in CvRh (R82 in BR) was discovered as a critical residue in the proton release complex whereas the residues E193 and E203 (194 and 204 in BR) are of minor relevance. Replacement of R83 by Q drastically reduced the power of the pump and caused outward directed pumping at neutral pH but inward directed passive currents at high load. We identified the proton donor Glu (or Asp) that reprotonates the retinal Schiff base and the proton shuttle Arg that transports the proton from the Retinal Schiff base counter ion (D85 in BR) to the extracellular side as the key determinants for the directivity and the power of the pumps.

Chromatin dynamics at the hTERT promoter during transcriptional activation and repression by c-Myc and Mnt in Xenopus leavis oocytes

The transcription factors c-Myc and Mnt regulate gene expression through dimerization with Max and binding to E-boxes in target genes. While c-Myc activates gene expression via recruitment of histone modifying complexes, Mnt acts as a transcriptional repressor. Here, we used the Xenopus leavis oocyte system to address the effect of c-Myc and Mnt on transcription and chromatin remodeling over the E-box region in the human telomerase reverse transcriptase (hTERT) promoter. As expected we found elevated and decreased levels of hTERT transcription upon exogenously expressed c-Myc/Max and Mnt/Max, respectively. In addition, we confirmed binding of these heterodimers to both E-boxes already enriched with H3K9ac and H4K16ac. These chromatin marks were further enhanced upon c-Myc/Max binding followed by increased DNA accessibility in the E-box region. In contrast, Mnt/Max inhibited Myc-induced transcription and mediated repression through complete chromatin condensation and deacetylation of H3K9 and H4K16 across the E-box region. Importantly, Mnt was able to counteract c-Myc mediated activation even when expressed at low levels, suggesting Mnt to act as a strong repressor by closing the chromatin structure. Collectively our data demonstrate that the balance between c-Myc and Mnt activity determines the transcriptional outcome of the hTERT promoter by modulation of the chromatin architecture.

The hZIP4 transporter has two binding affinities and transports multiple transition metal ions.

Cells possess numerous membrane proteins to transport essential metal ions across cellular organelles. hZIP4 (human ZIP4) is one member of the ZIP (Zrt‐ and Irt‐like proteins) family of proteins that imports zinc to the cytoplasm. Autosomal mutations in hZIP4 have been associated with the zinc deficient genetic disorder Acrodermatitis Enteropathica. hZIP4 is expressed in small intestine, stomach, colon, cecum, kidney and pancreas. Recently, hZIP4 overexpression has also been associated with the pancreatic cancer. hZIP4 has 8 transmembrane domains with extracellular N‐ and C‐termini. Furthermore, the histidine rich non‐conserved N‐terminus and intracellular domain between transmembrane‐III and IV are believed to be important for Zn2+ coordination.We have established a radio‐isotope uptake experiment in Xenopus leavis oocytes to study the selectivity and kinetics of hZIP4 cation transport. An analysis of our experimental results demonstrate that hZIP4 transports Ni2+ and Cu2+ in addition to Zn2+. Our data also shows that hZIP4 has two binding affinities where one is in the μM range and the other one is in nM concentration. Subsequent experiments have also identified possible zinc coordination amino acid residues.

Clobazam and Its Active Metabolite, N-Desmethylclobazam, Are Partial Benzodiazepine Receptor Agonists at Cloned GABAA Receptors Expressed in Xenopus leavis Oocytes (P05.085)

Objective: We investigated functional effects of clobazam and its active metabolite, N-desmethylclobazam (N-CLB), on recombinant GABA A receptors by electrophysiology. Background Clobazam, a 1,5-benzodiazepine, is in FDA review for Lennox-Gastaut syndrome. Design/Methods: To better understand the observed clinical profile of clobazam, we first characterized its GABA A -receptor-subtype–dependent positive allosteric modulation. Using two electrode voltage clamps, we determined that clobazam and N-CLB were both positive allosteric modulators at Xenopus leavis oocytes injected with cRNA encoding different human GABA A -receptor subunits. GABA concentration-response curves were generated at combinations of α (α 1 , α 2 , α 3 , and α 5 ), β 2, and γ 2 subunits injected at 1:1:1 ratios. At each oocyte, maximum response to GABA and EC 50 and EC 20 values were detemined. Modulatory effects of the two 1,5-benzodiazepines to GABA EC 20 response was determined by co-application of different concentrations of the individual benzodiazepines with a GABA EC 20 concentration. Potentiation of GABA EC 20 response exerted by the respective benzodiazepines was related to maximum responses elicited by GABA on the GABA A -receptor–expressing oocyte. Results: Clobazam and N-CLB both acted as potent partial agonists at α 1 β 2 γ 2 and α 2 β 2 γ 2 receptors. Clobazam exhibited an EC 50 value of 120 nM and a maximum effect of 51% of that of the maximum response of GABA at α 1 β 2 γ 2 receptors, while it displayed an EC 50 value of 110 nM and a maximum effect of 50% at α 2 β 2 γ 2 receptors. Similarly, N-CLB displayed an EC 50 value of 130 nM and maximum effect of 39% at α 1 β 2 γ 2 , and an EC 50 value of 110 nM and maximum effect of 51% at α 2 β 2 γ 2 . Conclusions: Clobazam and N-desmethylclobazam were shown to be partial agonists acting through the benzodiazepine site of the GABA A receptor. This partial agonism may contribute to clobazam9s overall clinical effects and raises the possibility that clobazam9s tolerance profile in animal models may be different than those of 1,4-benzodiazepines such as diazepam. Supported by: H. Lundbeck A/S. Disclosure: Dr. Jensen has received research support from Lundbeck A/S. Dr. Hammer has received research support from Lundbeck Research USA, Inc. Dr. Ebert has received personal compensation for activities with Lundbeck Research USA as an employee. Dr. Sindal Jensen has received personal compensation for activities with H. Lundbeck A/S as an employee.

A Natural Point Mutation Reveals Target Promiscuity of Toxins Isolated from the Sea Anemone Anthopleura Elegantissima

Sea anemone venom is a known source of interesting bioactive compounds, including peptide toxins which are invaluable tools for studying structure and function of voltage-gated potassium channels. APETx3 is a novel peptide isolated from the sea anemone Anthopleura elegantissima, containing 42 amino acids cross-linked by 3 disulfide bridges. Sequence alignment reveals that APETx3 is a natural occurring mutant from APETx1, only differing in 1 amino acid at position 3. APETx1 is believed to be a selective modulator of human ether-a-go-go related (hERG) potassium channels. In this study, APETx1, 2 and 3 have been subjected to an electrophysiological screening on a wide range of 21 ion channels expressed in Xenopus leavis oocytes: 10 cloned voltage-gated sodium channels (NaV1.2-NaV1.8, the insect channels DmNaV1, BgNaV1-1a and the arachnid channel VdNaV1) and 11 cloned voltage-gated potassium channels (KV2.1, KV3.1, KV4.2, KV4.3, KV7.1, KV7.2, KV7.3, KV7.4, KV7.5, hERG, the insect channel Shaker IR). Surprisingly, the Thr3Pro substitution results in a complete abolishment of APETx3 modulation on hERG channels. However, the same substitution provides this toxin the ability to become a potent modulator of voltage-gated sodium channels (NaVs). APETx3 slows down the inactivation of mammalian and insect channels similar to site 3 toxins such as α-scorpion toxins and sea anemone NaVs toxins. Our screening reveals that the homologous toxins APETx1 and APETx2 display promiscuous properties as they are also capable of recognizing NaV channels, causing an inhibition of the sodium conductance.All together, these data provide new insights in key residues which allow these toxins to recognize distinct ion channels with similar potency but with different modulatory effects. Furthermore, we describe for the first time the target promiscuity of a family of sea anemone toxins believed to be highly selective.

Examining the Conformational Dynamics of Membrane Proteins <em>in situ</em> with Site-directed Fluorescence Labeling

Two electrode voltage clamp electrophysiology (TEVC) is a powerful tool to investigate the mechanism of ion transport1 for a wide variety of membrane proteins including ion channels2, ion pumps3, and transporters4. Recent developments have combined site-specific fluorophore labeling alongside TEVC to concurrently examine the conformational dynamics at specific residues and function of these proteins on the surface of single cells.We will describe a method to study the conformational dynamics of membrane proteins by simultaneously monitoring fluorescence and current changes using voltage-clamp fluorometry. This approach can be used to examine the molecular motion of membrane proteins site-specifically following cysteine replacement and site-directed fluorophore labeling5,6. Furthermore, this method provides an approach to determine distance constraints between specific residues7,8. This is achieved by selectively attaching donor and acceptor fluorophores to two mutated cysteine residues of interest.In brief, these experiments are performed following functional expression of the desired protein on the surface of Xenopus leavis oocytes. The large surface area of these oocytes enables facile functional measurements and a robust fluorescence signal5. It is also possible to readily change the extracellular conditions such as pH, ligand or cations/anions, which can provide further information on the mechanism of membrane proteins4. Finally, recent developments have also enabled the manipulation of select internal ions following co-expression with a second protein9.Our protocol is described in multiple parts. First, cysteine scanning mutagenesis proceeded by fluorophore labeling is completed at residues located at the interface of the transmembrane and extracellular domains. Subsequent experiments are designed to identify residues which demonstrate large changes in fluorescence intensity (<5%)3 upon a conformational change of the protein. Second, these changes in fluorescence intensity are compared to the kinetic parameters of the membrane protein in order to correlate the conformational dynamics to the function of the protein10. This enables a rigorous biophysical analysis of the molecular motion of the target protein. Lastly, two residues of the holoenzyme can be labeled with a donor and acceptor fluorophore in order to determine distance constraints using donor photodestruction methods. It is also possible to monitor the relative movement of protein subunits following labeling with a donor and acceptor fluorophore.

Examining the Conformational Dynamics of Membrane Proteins &lt;em&gt;in situ&lt;/em&gt; with Site-directed Fluorescence Labeling

Two electrode voltage clamp electrophysiology (TEVC) is a powerful tool to investigate the mechanism of ion transport1 for a wide variety of membrane proteins including ion channels2, ion pumps3, and transporters4. Recent developments have combined site-specific fluorophore labeling alongside TEVC to concurrently examine the conformational dynamics at specific residues and function of these proteins on the surface of single cells. We will describe a method to study the conformational dynamics of membrane proteins by simultaneously monitoring fluorescence and current changes using voltage-clamp fluorometry. This approach can be used to examine the molecular motion of membrane proteins site-specifically following cysteine replacement and site-directed fluorophore labeling5,6. Furthermore, this method provides an approach to determine distance constraints between specific residues7,8. This is achieved by selectively attaching donor and acceptor fluorophores to two mutated cysteine residues of interest. In brief, these experiments are performed following functional expression of the desired protein on the surface of Xenopus leavis oocytes. The large surface area of these oocytes enables facile functional measurements and a robust fluorescence signal5. It is also possible to readily change the extracellular conditions such as pH, ligand or cations/anions, which can provide further information on the mechanism of membrane proteins4. Finally, recent developments have also enabled the manipulation of select internal ions following co-expression with a second protein9. Our protocol is described in multiple parts. First, cysteine scanning mutagenesis proceeded by fluorophore labeling is completed at residues located at the interface of the transmembrane and extracellular domains. Subsequent experiments are designed to identify residues which demonstrate large changes in fluorescence intensity (<5%)3 upon a conformational change of the protein. Second, these changes in fluorescence intensity are compared to the kinetic parameters of the membrane protein in order to correlate the conformational dynamics to the function of the protein10. This enables a rigorous biophysical analysis of the molecular motion of the target protein. Lastly, two residues of the holoenzyme can be labeled with a donor and acceptor fluorophore in order to determine distance constraints using donor photodestruction methods. It is also possible to monitor the relative movement of protein subunits following labeling with a donor and acceptor fluorophore.

Revisiting the Heteromultimeric Structure of ENaC in XENOPUS Leavis Oocytes

The functional epithelial sodium channel (ENaC) is a heteromeric channel formed by three homologous alpha, beta and gamma subunits.Several functional and biochemical studies have provided evidence that the ENaC is a heterotetramer formed by 2alpha, 1beta and 1gamma subunits. Recently, a high-resolution crystal structure has been obtained from an ortholog of ENaC, the acid-sensing ion channel ASIC1, which showed a homotrimeric channel. This discrepancy between two channels of the same ion channel family, motivated us to revisit the subunit oligomerization of ENaC.His-tagged ENaC alpha, beta and gamma subunits were expressed in Xenopus leavis oocytes. The three ENaC subunits can be co-purified on Ni+2-NTA agarose beads in an alpha beta gamma-ENaC complex. On Western blot, the ENaC subunits showed typical post-translation modifications associated with a functional channel. Using differentially tagged ENaC subunits, we investigated whether the ENaC complex contains more than a single alpha, beta or gamma subunits. Two differentially tagged alpha subunits co-purified with beta and gamma subunits, indicating that ENaC is formed by more than one alpha subunit. The purified ENaC complex channel eluted on Sephadex G 200 column in a fraction corresponding to a molecular weight of 350 kDa, which was higher than expected for a alpha beta gamma-ENaC.These data confirm previous reports that the functional ENaC channel is a heterotetramer made of 2alpha 1beta, and 1gamma subunits.

Four and a Half Lim Domains (FHL) Genes Reduce Conductivity of the KCNA5 Channel

Myopathies are inherited muscle disorders characterized by weakness and atrophy of voluntary skeletal muscles, sometimes including the cardiac muscle. A phenotypically distinct, X-linked myopathy with postural muscle atrophy, termed XMPMA, has been recently described and linked to mutations in the FHL1 gene. FHL1, a member of LIM-only proteins, is expressed in skeletal and cardiac muscle and suggested to play a role in sarcomere synthesis and assembly. Three splice variants (A, B and C) exist, which differ in expression pattern, binding partners and subcellular localization. A mutation found in a large XMPMA family (C224W) affects only isoforms A and B.Aim of our study is to functionally characterize mutated FHL1 isoforms and their interaction with the voltage-gated potassium channel (KCNA5 or Kv1.5), which is involved in cardiac excitability. These interactions may partly explain the cardiac involvement within the clinical spectrum of XMPMA patients.K+ currents were recorded in Xenopus leavis oocytes injected with KCNA5 mRNA with or without coexpression of FHL1AWT, FHL1AC224W or FHL1C. Upon coexpression of all three FHL1 proteins, K+ current density was differently decreased, when compared to oocytes expressing KCNA5 alone. Kinetics of the channel was not affected. These results support the role of FHL1 as a key molecular component in regulation of expression of KCNA5. Future experiments will concentrate on colocalization and molecular interaction of FHL1 and KCNA5 in mammalian in vitro systems (HL1 cells).Support by Graz Medical University Ph.D. programme and County of Styria (GZ: A3-16.R-10/2009-112).

Cardiovascular profile of newly developed Diltiazem analogs

Many diltiazem related L-VDCC blockers were developed using a multidisciplinary approach. This current study was to investigate and compare diltiazem with to the newly developed compounds by mouse Langendorff-perfused heart, Ca2+-transients and on recombinant L-VDCC. Five particular compounds were selected by the ligand-based virtual screening procedure (LBVS) (5B, M2, M7, M8 and P1). Wild-type human heart and rabbit lung α1 subunits were expressed (combined with the regulatory α2δ and β3 subunits) in Xenopus leavis oocytes using a two-electrode voltage clamp technique. Diltiazem is a benzothiazepine Ca2+ channel blocker used clinically for its antihypertensive and antiarrhythmic effects. Previous radioligand binding assays revealed a complex interaction with the benzothiazepine binding sites for M2, M7 and M8. (Carosati E. et al. J. Med Chem. 2006, 49; 5206). In agreement with this, the relative order of increased rates of contraction and relaxation at lower concentrations ( M2>M8>P1. Similar increases in Ca2+ transients were observed in cardiomyocytes. Diltiazem showed negative inotropic effects whereas 5B had no significant effect. Diltiazem blocks Ca2+ currents in a use-dependent manner and facilitates the channel by accelerating the inactivation and decelerating the recovery from inactivation. In contrast to diltiazem, the new analogs had no pronounced use-dependence. Application of 100 μM M8 and M2 showed ∼10% tonic block, shifted the steady-state inactivation in hyperpolarized direction and the current inactivation time was significantly decreased compared with control (219.6±11.5 ms, 226±14.5 vs. 269±12.9 ms). Contrary to diltiazem, the recovery from the block by M8 and M2 was comparable to control. All of the compounds displayed the same sensitivity on the Ca2+ channel rabbit lung α1 except P1. Taken together, these findings suggest that M8 and M2 might directly decrease the binding affinity or allow more rapid dissociation from the benzothiazepine binding site.

Functional Characterization Of Clc-5 Mutations Associated With Dent's Disease

The Cl−/H+ antiporter ClC-5 has been linked to Dent's disease, an X-linked renal disease associated with low molecular weight proteinuria, hypercalciuria and nephrolithiasis. ClC-5 is expressed on early endosomes of proximal tubule cells, where it plays a critical role in endosomal function. The impact of Dent's disease-causing mutations on ClC-5 function has not been yet fully investigated. Here, we have analysed an unpublished mutation K115R and three published mutations, Y272C, N340K and K546E in terms of electrical activity and trafficking at the plasma membrane in Xenopus leavis oocytes.

A fruit‐specific plasma membrane aquaporin subtype PIP1;1 is regulated during strawberry (Fragaria × ananassa) fruit ripening

Despite the advances in the physiology of fruit ripening, the role and contribution of water pathways are still barely considered. Our aim was therefore to characterize aquaporins, proteins that render the molecular basis for putative regulatory mechanisms in water transport. We focused our work on strawberry (Fragaria xananassa) fruit, a non-climacteric fruit of special interest because of its forced brief commercial shelf life. A full-length cDNA was isolated with high homology with plasma membrane (PM) intrinsic proteins (named FaPIP1;1), showing a profile with high expression in fruit, less in ovaries and no detection at all in other parts. Its cellular localization was confirmed at the PM. As reported in other plasma membrane intrinsic proteins subtype 1 (PIP1s), when expressing the protein in Xenopus leavis oocytes, FaPIP1;1 shows low water permeability values that only increased when it is coexpressed with a plasma membrane intrinsic protein subtype 2. Northern blotting using total RNA shows that its expression increases during fruit ripening. Moreover, functional characterization of isolated PM vesicles from red stage fruit unequivocally demonstrates the presence of active water channels, i.e. high water permeability values and a low Arrhenius activation energy, both evidences of water transport mediated by proteins. Interestingly, as many ripening-related strawberry genes, the expression pattern of FaPIP1;1 was also repressed by the presence of auxins. We therefore report a fruit specific PIP1 aquaporin with an accumulation pattern tightly associated to auxins and to the ripening process that might be responsible for increasing water permeability at the level of the PM in ripe fruit.