ABSTRACT␣4 and ␤2 nicotinic acetylcholine receptor (nAChR) subunits expressed heterologously assemble into receptors with high (HS) and low (LS) sensitivity to acetylcholine (ACh); their relative proportions depend on the ␣4 to ␤2 ratio. In this study, injection of oocytes with 1:10 ␣4/␤2 subunit cDNA ratios favored expression of HS ␣4␤2 nAChRs, as evidenced by monophasic ACh concentration-response curves, whereas injections with 10:1 cDNA ratios favored expression of LS ␣4␤2 receptors. The stoichiometry was inferred from the shifts in the ACh EC 50 values caused by Leu to Thr mutations at position 9Ј of the second transmembrane domain of ␣4 and ␤2. The 1:10 injection ratio produced the (␣4) 2 (␤2) 3 stoichiometry, whereas 10:1 injections produced the (

The joints of mammals are lined with cartilage, comprised of individual chondrocytes embedded in a specialized extracellular matrix. Chondrocytes experience a complex mechanical environment and respond to changing mechanical loads in order to maintain cartilage homeostasis. It has been proposed that mechanically gated ion channels are of functional importance in chondrocyte mechanotransduction; however, direct evidence of mechanical current activation in these cells has been lacking. We have used high-speed pressure clamp and elastomeric pillar arrays to apply distinct mechanical stimuli to primary murine chondrocytes, stretch of the membrane and deflection of cell-substrate contacts points, respectively. Both TRPV4 and PIEZO1 channels contribute to currents activated by stimuli applied at cell-substrate contacts but only PIEZO1 mediates stretch-activated currents. These data demonstrate that there are separate, but overlapping, mechanoelectrical transduction pathways in chondrocytes.DOI: http://dx.doi.org/10.7554/eLife.21074.001

Nicotinic acetylcholine receptor (nAChR) ␣4 and ␤2 subunits assemble in two alternate stoichiometries to produce (␣4␤2) 2 ␣4 and (␣4␤2) 2 ␤2, which display different agonist sensitivities. Functionally relevant agonist binding sites are thought to be located at ␣4(؉)/␤2(؊) subunit interfaces, but because these interfaces are present in both receptor isoforms, it is unlikely that they account for differences in agonist sensitivities. In contrast, incorporation of either ␣4 or ␤2 as auxiliary subunits produces isoform-specific ␣4(؉)/␣4(؊) or ␤2(؉)/␤2(؊) interfaces. Using fully concatenated (␣4␤2) 2 ␣4 nAChRs in conjunction with structural modeling, chimeric receptors, and functional mutagenesis, we have identified an additional site at the ␣4(؉)/ ␣4(؊) interface that accounts for isoform-specific agonist sensitivity of the (␣4␤2) 2 ␣4 nAChR. The additional site resides in a region that also contains a potentiating Zn 2؉ site but is engaged by agonists to contribute to receptor activation. By engineering ␣4 subunits to provide a free cysteine in loop C at the ␣4(؉)␣4(؊) interface, we demonstrated that the acetylcholine responses of the mutated receptors are attenuated or enhanced, respectively, following treatment with the sulfhydryl reagent [2-(trimethylammonium)ethyl]methanethiosulfonate or aminoethyl methanethiosulfonate. The findings suggest that agonist occupation of the site at the ␣4(؉)/(␣4(؊) interface leads to channel gating through a coupling mechanism involving loop C. Overall, we propose that the additional agonist site at the ␣4(؉)/ ␣4(؊) interface, when occupied by agonist, contributes to receptor activation and that this additional contribution underlies the agonist sensitivity signature of (␣4␤2) 2 ␣4 nAChRs.

Background and purpose: a4 and b2 nicotinic acetylcholine (ACh) receptor subunits expressed heterologously in Xenopus oocytes assemble into a mixed population of (a4)2(b2)3 and (a4)3(b2)2 receptors. In order to express these receptors separately in heterologous systems, we have engineered pentameric concatenated (a4)2(b2)3 and (a4)3(b2)2 receptors. Experimental approach: a4 and b2 subunits were concatenated by synthetic linkers into pentameric constructs to produce either (a4)2(b2)3 or (a4)3(b2)2 receptors. Using two-electrode voltage-clamp techniques, we examined the ability of the concatenated constructs to produce functional expression in Xenopus oocytes. Functional constructs were further characterized in respect to agonists, competitive antagonists, Ca 2+ permeability, sensitivity to modulation by Zn 2+ and sensitivity to up-regulation by chaperone protein 14-3-3. Key results: We found that pentameric concatamers with a subunit arrangement of b2_a4_b2_a4_b2 or b2_a4_b2_a4_a4 were stable and functional in Xenopus oocytes. By comparison, when a4 and b2 were concatenated with a subunit order of b2_b2_a4_b2_a4 or b2_a4_a4_b2_a4, functional expression in Xenopus oocytes was very low, even though the proteins were synthesized and stable. Both b2_a4_b2_a4_b2 and b2_a4_b2_a4_a4 concatamers recapitulated the ACh concentration response curve, the sensitivity to Zn 2+ modulation, Ca 2+ permeability and the sensitivity to up-regulation by chaperone protein 14-3-3 of the corresponding non-linked (a4)2(b2)3 and (a4)3(b2)2 receptors respectively. Using these concatamers, we found that most a4b2-preferring compounds studied, including A85380, 5I-A85380, cytisine, epibatidine, TC2559 and dihydro-berythroidine, demonstrate stoichiometry-specific potencies and efficacies. Conclusions and implications:We concluded that the a4b2 nicotinic ACh receptors produced with b2_a4_b2_a4_b2 or b2_a4_b2_a4_a4 pentameric constructs are valid models of non-linked (a4)2(b2)3 and (a4)3(b2)2 receptors respectively.

Fatty acids (FAs) are not only essential components of cellular energy storage and structure, but play crucial roles in signalling. Here we present a toolkit of photoswitchable FA analogues (FAAzos) that incorporate an azobenzene photoswitch along the FA chain. By modifying the FAAzos to resemble capsaicin, we prepare a series of photolipids targeting the Vanilloid Receptor 1 (TRPV1), a non-selective cation channel known for its role in nociception. Several azo-capsaicin derivatives (AzCAs) emerge as photoswitchable agonists of TRPV1 that are relatively inactive in the dark and become active on irradiation with ultraviolet-A light. This effect can be rapidly reversed by irradiation with blue light and permits the robust optical control of dorsal root ganglion neurons and C-fibre nociceptors with precision timing and kinetics not available with any other technique. More generally, we expect that photolipids will find many applications in controlling biological pathways that rely on protein–lipid interactions.