Mohamed Kreir

Human TRPA1 is intrinsically cold- and chemosensitive with and without its N-terminal ankyrin repeat domain

Significance The ability of an organism to detect and avoid noxious temperatures is crucial for survival. It is, therefore, of great interest that several transient receptor potential (TRP) ion channels have been proposed as temperature sensors. However, to date, only the menthol receptor (TRP subtype M8) and the chili pepper receptor (TRP subtype V1) have been shown to be intrinsically temperature-sensitive proteins in mammals. In this study, we show that the purified wasabi receptor (TRP subtype A1) is a cold sensor. Thus, mammals have at least two cold sensors that, together, cover pleasant (TRP subtype M8) and unpleasant (TRP subtype A1) cold temperatures. Our findings add to the understanding of how the temperature sense is organized and its role in pain associated with cold hypersensitivity. We have purified and reconstituted human transient receptor potential (TRP) subtype A1 (hTRPA1) into lipid bilayers and recorded single-channel currents to understand its inherent thermo- and chemosensory properties as well as the role of the ankyrin repeat domain (ARD) of the N terminus in channel behavior. We report that hTRPA1 with and without its N-terminal ARD (Δ1–688 hTRPA1) is intrinsically cold-sensitive, and thus, cold-sensing properties of hTRPA1 reside outside the N-terminal ARD. We show activation of hTRPA1 by the thiol oxidant 2-((biotinoyl)amino)ethyl methanethiosulfonate (MTSEA-biotin) and that electrophilic compounds activate hTRPA1 in the presence and absence of the N-terminal ARD. The nonelectrophilic compounds menthol and the cannabinoid Δ9-tetrahydrocannabiorcol (C16) directly activate hTRPA1 at different sites independent of the N-terminal ARD. The TRPA1 antagonist HC030031 inhibited cold and chemical activation of hTRPA1 and Δ1–688 hTRPA1, supporting a direct interaction with hTRPA1 outside the N-terminal ARD. These findings show that hTRPA1 is an intrinsically cold- and chemosensitive ion channel. Thus, second messengers, including Ca2+, or accessory proteins are not needed for hTRPA1 responses to cold or chemical activators. We suggest that conformational changes outside the N-terminal ARD by cold, electrophiles, and nonelectrophiles are important in hTRPA1 channel gating and that targeting chemical interaction sites outside the N-terminal ARD provides possibilities to fine tune TRPA1-based drug therapies (e.g., for treatment of pain associated with cold hypersensitivity and cardiovascular disease).

Voltage-gated sodium channel (NaV) protein dissection creates a set of functional pore-only proteins

Many voltage-gated ion channel (VGIC) superfamily members contain six-transmembrane segments in which the first four form a voltage-sensing domain (VSD) and the last two form the pore domain (PD). Studies of potassium channels from the VGIC superfamily together with identification of voltage-sensor only proteins have suggested that the VSD and the PD can fold independently. Whether such transmembrane modularity is common to other VGIC superfamily members has remained untested. Here we show, using protein dissection, that the Silicibacter pomeroyi voltage-gated sodium channel (NaVSp1) PD forms a stand-alone, ion selective pore (NaVSp1p) that is tetrameric, α-helical, and that forms functional, sodium-selective channels when reconstituted into lipid bilayers. Mutation of the NaVSp1p selectivity filter from LESWSM to LDDWSD, a change similar to that previously shown to alter ion selectivity of the bacterial sodium channel NaVBh1 (NaChBac), creates a calcium-selective pore-only channel, CaVSp1p. We further show that production of PDs can be generalized by making pore-only proteins from two other extremophile NaVs: one from the hydrocarbon degrader Alcanivorax borkumensis (NaVAb1p), and one from the arsenite oxidizer Alkalilimnicola ehrlichei (NaVAe1p). Together, our data establish a family of active pore-only ion channels that should be excellent model systems for study of the factors that govern both sodium and calcium selectivity and permeability. Further, our findings suggest that similar dissection approaches may be applicable to a wide range of VGICs and, thus, serve as a means to simplify and accelerate biophysical, structural, and drug development efforts.

Port-a-patch and patchliner: high fidelity electrophysiology for secondary screening and safety pharmacology.

Ion channel dysfunction is known to underlie several acute and chronic disorders and, therefore, ion channels have gained increased interest as drug targets. During the past decade, ion channel screening platforms have surfaced that enable high throughput drug screening from a more functional perspective. These two factors taken together have further inspired the development of more refined screening platforms, such as the automated patch clamp platforms described in this article. Approximately six years ago, Nanion introduced its entry level device for automated patch clamping - the Port-a-Patch. With this device, Nanion offers the worlds smallest patch-clamp workstation, whilst greatly simplifying the experimental procedures. This makes the patch clamp technique accessible to researchers and technicians regardless of previous experience in electrophysiology. The same flexibility and high data quality is achieved in a fully automated manner with the Patchliner, Nanions higher throughput patch clamp workstation. The system utilizes a robotic liquid handling environment for fully automated application of solutions, cells and compounds. The NPC-16 chips come in a sophisticated, yet simplistic, microfluidic cartridge, which allow for fast and precise perfusion. In this way, full concentration response curves are easily obtained. The Port-a-Patch and Patchliner workstations from Nanion are valuable tools for target validation, secondary screening and safety pharmacology (for example hERG and Nav1.5 safety screening). They are widely used in drug development efforts by biotechnological and pharmaceutical companies, as well as in basic and applied biophysical research within academia.

Permeation of antibiotics through Escherichia coli OmpF and OmpC porins: screening for influx on a single-molecule level.

A chip-based automated patch-clamp technique provides an attractive biophysical tool to quantify solute permeation through membrane channels. Proteo–giant unilamellar vesicles (proteo-GUVs) were used to form a stable lipid bilayer across a micrometer-sized hole. Because of the small size and hence low capacitance of the bilayer, single-channel recordings were achieved with very low background noise. The latter allowed the characterization of the influx of 2 major classes of antibiotics—cephalosporins and fluoroquinolones—through the major Escherichia coli porins OmpF and OmpC. Analyzing the ion current fluctuations in the presence of antibiotics revealed transport properties that allowed the authors to determine the mode of permeation. The chip-based setup allows rapid solution exchange and efficient quantification of antibiotic permeation through bacterial porins on a single-molecule level.

Planar patch clamp: advances in electrophysiology.

Ion channels have gained increased interest as therapeutic targets over recent years, since a growing number of human and animal diseases have been attributed to defects in ion channel function. Potassium channels are the largest and most diverse family of ion channels. Pharmaceutical agents such as Glibenclamide, an inhibitor of K(ATP) channel activity which promotes insulin release, have been successfully sold on the market for many years. So far, only a small group of the known ion channels have been addressed as potential drug targets. The functional testing of drugs on these ion channels has always been the bottleneck in the development of these types of pharmaceutical compounds.New generations of automated patch clamp screening platforms allow a higher throughput for drug testing and widen this bottleneck. Due to their planar chip design not only is a higher throughput achieved, but new applications have also become possible. One of the advantages of planar patch clamp is the possibility of perfusing the intracellular side of the membrane during a patch clamp experiment in the whole-cell configuration. Furthermore, the extracellular membrane remains accessible for compound application during the experiment.Internal perfusion can be used not only for patch clamp experiments with cell membranes, but also for those with artificial lipid bilayers. In this chapter we describe how internal perfusion can be applied to potassium channels expressed in Jurkat cells, and to Gramicidin channels reconstituted in a lipid bilayer.

Membrane assembly of the functional KcsA potassium channel in a vesicle-based eukaryotic cell-free translation system.

The potassium channel KcsA was heterologously expressed in a eukaryotic cell-free system. Both, the expression yields and functional analysis of the protein were reported. Qualitative and quantitative analyses of KcsA expression were performed by using (14)C-labeled leucine as one of the amino acids supplemented in the cell-free reaction mixture. There was a time dependent increase in the protein yield as well as the intensity of the native tetramer band in insect cell derived microsomes. Electrophysiology measurements demonstrated the functional activity of the microsomes harboring KcsA showing single-channel currents with the typical biophysical characteristics of the ion channel. The channel behavior was asymmetric and showed positive rectification with larger currents towards positive voltages. KcsA channel currents were effectively blocked by potassium selective barium (Ba(2+)). This functional demonstration of an ion channel in eukaryotic cell-free system has a large potential for future applications including drug screening, diagnostic applications and functional assessment of complex membrane proteins like GPCRs by coupling them to ion channels in cell-free systems. Furthermore, membrane proteins can be expressed directly from linear DNA templates within 90 min, eliminating the need for additional cloning steps, which makes this cell-free system fast and efficient.

Green Fluorescent Protein Changes the Conductance of Connexin 43 (Cx43) Hemichannels Reconstituted in Planar Lipid Bilayers

Background: Connexin 43 hemichannels participate in many cellular processes. To elucidate their location and function within living cells, they were labeled with GFP. Results: Recombinantly expressed Cx43 and Cx43-GFP form conducting hemichannels in reconstituted planar membranes. Their conductance states and voltage dependence differ. Conclusion: Fusion of GFP to Cx43 significantly affects the electrophysiological behavior of Cx43 hemichannels. Significance: GFP can significantly alter channel activities. In mammalian tissues, connexin 43 (Cx43) is the most prominent member of the connexin family. In a single lipid bilayer, six connexin subunits assemble into a hemichannel (connexon). Direct communication of apposing cells is realized by two adjacent hemichannels, which can form gap junction channels. Here, we established an expression system in Pichia pastoris to recombinantly produce and purify Cx43 as well as Cx43 fused to green fluorescent protein (GFP). Proteins were isolated from crude cell membrane fractions via affinity chromatography. Cx43 and Cx43-GFP hemichannels were reconstituted in giant unilamellar vesicles as proven by fluorescence microscopy, and their electrophysiological behavior was analyzed on the single channel level by planar patch clamping. Cx43 and Cx43-GFP both showed an ohmic behavior and a voltage-dependent open probability. Cx43 hemichannels exhibited one major mean conductance of 224 ± 26 picosiemens (pS). In addition, a subconductance state at 124 ± 5 pS was identified. In contrast, the analysis of Cx43-GFP single channels revealed 10 distinct conductance states in the range of 15 to 250 pS, with a larger open probability at 0 mV as compared with Cx43, which suggests that intermolecular interactions between the GFP molecules alter the electrophysiology of the protein.

Different ligands of the TRPV3 cation channel cause distinct conformational changes as revealed by intrinsic tryptophan fluorescence quenching.

Background: Further insight into the structural biology of TRP channels is crucial to explain molecular mechanisms of channel function. Results: We purified TRPV3, demonstrated its functional integrity, and used fluorescence spectroscopy to study ligand binding. Conclusion: TRPV3 ligands induce different conformational changes as observed by tryptophan fluorescence quenching. Significance: Availability of purified TRPV3 allows functional assays outside the cellular context and facilitates future structural studies. TRPV3 is a thermosensitive ion channel primarily expressed in epithelial tissues of the skin, nose, and tongue. The channel has been implicated in environmental thermosensation, hyperalgesia in inflamed tissues, skin sensitization, and hair growth. Although transient receptor potential (TRP) channel research has vastly increased our understanding of the physiological mechanisms of nociception and thermosensation, the molecular mechanics of these ion channels are still largely elusive. In order to better comprehend the functional properties and the mechanism of action in TRP channels, high-resolution three-dimensional structures are indispensable, because they will yield the necessary insights into architectural intimacies at the atomic level. However, structural studies of membrane proteins are currently hampered by difficulties in protein purification and in establishing suitable crystallization conditions. In this report, we present a novel protocol for the purification of membrane proteins, which takes advantage of a C-terminal GFP fusion. Using this protocol, we purified human TRPV3. We show that the purified protein is a fully functional ion channel with properties akin to the native channel using planar patch clamp on reconstituted channels and intrinsic tryptophan fluorescence spectroscopy. Using intrinsic tryptophan fluorescence spectroscopy, we reveal clear distinctions in the molecular interaction of different ligands with the channel. Altogether, this study provides powerful tools to broaden our understanding of ligand interaction with TRPV channels, and the availability of purified human TRPV3 opens up perspectives for further structural and functional studies.

Ion Channel Reconstitution

Since the discovery of the possible solubilization of membrane proteins and their isolation from other membrane constituents (purification), different methods were developed to reconstitute ion channel proteins into artificial lipid bilayers. These membrane proteins were then fully functional when correctly oriented and inserted in a lipid bilayer. The reconstitution plays a central role in identifying and characterizing the mechanisms of action of membrane proteins. The activity of the membrane proteins is studied using electrophysiology using different methods e.g., the black lipid membrane. Therefore, the structure-function relationship can be investigated to better understand the biophysical properties of membrane proteins in vivo.Using a glass surface containing a micrometer hole, the fusion of vesicles on the surface becomes an attractive method for electrophysiology and then to reconstitute membrane proteins into the lipid bilayer without denaturation. Then stable lipid bilayers are formed by bursting a GUV on the glass surface, forming a free-standing portion above the hole.With this technique, we studied the biophysical and pharmacological properties of different ion channels, for example potassium channels (KcsA, Kv1.2), sodium channels (NachBac, NaVsp1) as well as other ligand-dependent (IP3 receptor, NMDA receptor), mechanosensitive channels (MscL, TRP channels) and non-specific channels (Cx43, VDAC). I will describe here our methods for incorporation of proteins into the bilayer and the recording of single ion channel current measurements by using a planar patch clamp platform.

Single Hemichannels Recorded in Lipid Bilayers and Artificial Gap Junction Formation with Cells

Connexins (Cx) are members of a multigene family of membrane-spanning proteins that form gap junctions, which are composed of two hexameric hemichannels, called connexons. These gap junctions, organized in so-called gap junctional plaques, span the extracellular space/matrix of adjacent cells and thus allow a passive exchange of small molecules up to about 1 kDa. Connexins are widely distributed with various subtypes of connexin and are involved in different biological processes such transmission of information and propagation of action potential for e.g. Recent studies indicates that hemichannels do open under physiological and pathological conditions.In our study, we investigated the biophysical properties of hemichannels Cx26 and Cx43 which were isolated biochemically and reconstituted into synthetic lipid membranes. Both hemichannels are present in different tissues and involved in different pathologies. The results on a study of the Cx26 are presented. Reconstitutions of functional Cx26 and mutant hemichannels were performed. Secondly, Cx43 was purified and reconstituted into bilayers. The hemichannel Cx43 properties were compared to previous studies and showed similarities of conductance on single channel recordings of Cx43 in cells. Our focus was then to form artificial gap junctions, first between two unrelated cells and then between cells and bilayers containing functional hemichannels. This was done using Cx26 or Cx43. The bilayer-cell configuration allows to measure electrophysiological properties of the cells indirectly via gap junctions. Single channel recordings of gap junctions were recorded using a bilayer containing Cx43 and Cardiomyocytes expressing Cx43. Macroscopic currents were as well recorded between bilayers and cell lines expressing Cx26 or Cx43.