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 Δ 9 -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 Ca 2+ , 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).cold sensing | irritants | pain | sensory neuron | TRP channels A number of vertebrate and invertebrate transient receptor potential (TRP) ion channels have been implicated in temperature sensation (1-3), but only the rat menthol receptor TRP subtype M8 (TRPM8) and the rat capsaicin receptor TRP subtype V1 (TRPV1) have been shown to possess intrinsic thermosensitivity (4, 5). In 2003, Story et al. (6) proposed that the mouse TRPA1 is a noxious cold sensor. Story et al. (6) showed that TRPA1 was present in nociceptive primary sensory neurons and that CHO cells heterologously expressing the mouse TRPA1 displayed cold sensitivity. Most subsequent studies of cold responses in heterologous TRPA1 expression systems, isolated primary sensory neurons, and whole animals have provided evidence in support of mouse and rat TRPA1 being involved in noxious cold transduction (7). Interestingly, a familial episodic pain syndrome triggered by cold is caused by a gain-of-function mutation in the TRPA1 gene, indicating that TRPA1 may have a key role in human noxious cold sensation (8). Thus, human TRPA1 (hTRPA1) may be a relevant drug target for treatment of this condition and other pathological conditions, such as inflammation, nerve injury, and chemotherapy-induced neuropathy, that are characterized by TRPA1-dependent cold allodynia or hypersensitivity (7). However, in vitro studies of the expressed hTRPA1 h...

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 voltagegated sodium channel (Na V Sp1) PD forms a stand-alone, ion selective pore (Na V Sp1p) that is tetrameric, α-helical, and that forms functional, sodium-selective channels when reconstituted into lipid bilayers. Mutation of the Na V Sp1p selectivity filter from LESWSM to LDDWSD, a change similar to that previously shown to alter ion selectivity of the bacterial sodium channel Na V Bh1 (NaChBac), creates a calcium-selective pore-only channel, Ca V Sp1p. We further show that production of PDs can be generalized by making pore-only proteins from two other extremophile Na V s: one from the hydrocarbon degrader Alcanivorax borkumensis (Na V Ab1p), and one from the arsenite oxidizer Alkalilimnicola ehrlichei (Na V Ae1p). 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.V oltage-gated sodium channels (Na V s) are large polytopic membrane proteins involved in action potential generation in excitable cells and belong to an ion channel superfamily that includes voltage-gated calcium channels (Ca V s), voltage-gated potassium channels (K V s), and transient receptor potential (TRP) channels (1, 2). Within the voltage-gated ion channel (VGIC) superfamily, Na V s and Ca V s are close relatives (1-3) that share a topology of 24 transmembrane segments organized in four homologous six-transmembrane repeats. These two families are also thought to share some common structure in the ion selectivity filter despite having markedly different ion permeation properties (4). Both are central to human neuromuscular, cardiovascular, and neural physiology. Consequently, they are targets for a host of pharmaceuticals used to treat a diverse set of disorders and remain active targets for drug development (5-7). Recently, single subunit, six-transmembrane segment Na V s have been identified in a large number of bacteria from diverse environments (8, 9). These channels show clear similarities to eukaryotic Na V s and Ca V s (2, 9, 10), suggesting that the prokaryotic channels may have been ancestors of the more complex vertebrate channels.Despite the central importance of Na V s, nothing is known about the high-resolution structure o...

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Solvent-free planar lipid bilayers were formed in an automatic manner by bursting of giant unilamellar vesicles (GUVs) after gentle suction application through micron-sized apertures in a borosilicate glass substrate. Incubation of GUVs with the purified ion channel protein of interest yielded proteoliposomes. These proteoliposomes allow for immediate recording of channel activity after GUV sealing. This approach reduces the time-consuming, laborious and sometimes difficult protein reconstitution processes normally performed after bilayer formation. Bilayer recordings are attractive for investigations of membrane proteins not accessible to patch clamp analysis, like e.g. proteins from organelles. In the presented work, we show the example of the outer membrane protein OmpF from Escherichia coli. We reconstituted OmpF in proteoliposomes and observed the characteristic trimeric conductance levels and the typical gating induced by pH and transmembrane voltage. Moreover, OmpF is the main entrance for beta-lactam antibiotics and we investigated translocation processes of antibiotics and modulation of OmpF by spermine. We suggest that the rapid formation of porin containing lipid bilayers is of potential for the efficient electrophysiological characterization of the OmpF protein, for studying membrane permeation processes and for the rapid screening of antibiotics.

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