Although it has been known for some time that olfactory receptors (ORs) reside in spermatozoa, the function of these ORs is unknown. Here, we identified, cloned, and functionally expressed a previously undescribed human testicular OR, hOR17-4. With the use of ratiofluorometric imaging, Ca2+ signals were induced by a small subset of applied chemical stimuli, establishing the molecular receptive fields for the recombinantly expressed receptor in human embryonic kidney (HEK) 293 cells and the native receptor in human spermatozoa. Bourgeonal was a powerful agonist for both recombinant and native receptor types, as well as a strong chemoattractant in subsequent behavioral bioassays. In contrast, undecanal was a potent OR antagonist to bourgeonal and related compounds. Taken together, these results indicate that hOR17-4 functions in human sperm chemotaxis and may be a critical component of the fertilization process.

Members of the superfamily of transient receptor potential (TRP) channels are proposed to play important roles in sensory physiology. As an excitatory ion channel TRPA1 is robustly activated by pungent irritants in mustard and garlic and is suggested to mediate the inflammatory actions of environmental irritants and proalgesic agents. Here, we demonstrate that, in addition to pungent natural compounds, Ca 2؉ directly gates heterologously expressed TRPA1 in whole-cell and excised-patch recordings with an apparent EC 50 of 905 nM. Pharmacological experiments and site-directed mutagenesis indicate that the N-terminal EFhand calcium-binding domain of the channel is involved in Ca 2؉ -dependent activation. Furthermore, we determine Ca 2؉ as prerequisite for icilin activity on TRPA1. The transient receptor potential channel A1 (TRPA1)2 is a member of the superfamily of TRP channels. In mammals, A1 forms its own subfamily and is distinguished from other TRP channels by the presence of ϳ14 ankyrin repeats in its N terminus (1). TRPA1 was initially described as a cold sensitive nonselective cation channel (2), but it also functions as a ligand-gated channel in heterologous expression systems and sensory neurons (3). The pungent ingredients in mustard (allyl isothiocyanate, AITC) and garlic (allicin) robustly activate TRPA1 currents (4 -6). In addition, TRPA1 appears to be regulated by phospholipase C (PLC)-coupled receptors, suggesting that channel opening can be mediated by second messengers (4,7,8).TRPA1 expression was first described in a subset of sensory neurons of dorsal root and trigeminal ganglia that contribute to nociception and co-express calcitonin gene related peptide, substance P and TRPV1 (2, 4). Recent studies on TRPA1 deficient mice support a role of the channel in inflammatory pain and sensation of noxious cold (8,9). A model suggests TRPA1 activation by bradykinin, a potent algogenic substance released due to tissue injury and inflammation, in two possible ways: through PLC-mediated increases in intracellular Ca 2ϩ or other metabolites (e.g. diacylglycerol) and via Ca 2ϩ influx through TRPV1 (9). Whether an increase in intracellular Ca 2ϩ is sufficient to activate TRPA1 is still debated (4, 7), but several findings indicate a role of Ca 2ϩ on TRPA1 function. It was shown that extracellular Ca 2ϩ enhances the current rate and magnitude of AITC-induced currents (4, 10). Furthermore, Ca 2ϩ is thought to be responsible for fast channel closure (10). In addition, single channel recordings of heterologously expressed TRPA1 revealed an AITC-induced conductance, which is reduced in the presence of Ca 2ϩ (10). Together these reports emphasize the importance of Ca 2ϩ for TRPA1 function. Very recently the existence of a putative EF-hand calciumbinding domain (EF-hand CBD) at the N terminus of TRPA1 was reported (11). The EF-hand CBD is the most common motif among Ca 2ϩ -binding sites of a large number of Ca 2ϩ -interacting proteins (12). The classical EF-hand is a helix-loophelix motif that coordinates the Ca 2ϩ...

Here, we provide the first evidence for functional expression of a human olfactory receptor protein (OR17-40) and show that recombinant olfactory receptors can be functionally expressed in heterologous systems. A mixture of 100 different odorants (Henkel 100) elicited a transient increase in intracellular [Ca(2+)] in human embryonic kidney 293 (HEK293) cells stably or transiently transfected with the plasmid pOR17-40. By subdividing the odorant mixture into progressively smaller groups, we identified a single component that represented the only effective substance: helional. Only the structurally closely related molecule heliotroplyacetone also activated the receptor. Other compounds, including piperonal, safrole, and vanillin, were completely ineffective. Mock-transfected cells and cells transfected with other receptors showed no change in intracellular [Ca(2+)] in response to odor stimulation. We were also able to functionally express OR17-40 in Xenopus laevis oocytes. Coexpression of a "reporter" channel allowed measurement of the response of oocytes injected with the cRNA of the human receptor to the odor mixture Henkel 100. The effective substances were the same (helional, heliotropylacetone) as those identified by functionally expressing the receptor in HEK293 cells and were active at the same, lower micromolar concentration. These findings open the possibility of now characterizing the sensitivity and specificity of many, if not all, of the hundreds of different human olfactory receptors.

No abstract

In response to cell swelling, volume-regulated anion channels (VRACs) participate in a process known as regulatory volume decrease (RVD). Only recently, first insight into the molecular identity of mammalian VRACs was obtained by the discovery of the leucine-rich repeats containing 8A (LRRC8A) gene. Here, we show that bestrophin 1 (BEST1) but not LRRC8A is crucial for volume regulation in human retinal pigment epithelium (RPE) cells. Whole-cell patch-clamp recordings in RPE derived from human-induced pluripotent stem cells (hiPSC) exhibit an outwardly rectifying chloride current with characteristic functional properties of VRACs. This current is severely reduced in hiPSC-RPE cells derived from macular dystrophy patients with pathologic BEST1 mutations. Disruption of the orthologous mouse gene (Best1 −/− ) does not result in obvious retinal pathology but leads to a severe subfertility phenotype in agreement with minor endogenous expression of Best1 in murine RPE but highly abundant expression in mouse testis. Sperm from Best1 −/− mice showed reduced motility and abnormal sperm morphology, indicating an inability in RVD. Together, our data suggest that the molecular identity of VRACs is more complex-that is, instead of a single ubiquitous channel, VRACs could be formed by cell type-or tissue-specific subunit composition. Our findings provide the basis to further examine VRAC diversity in normal and diseased cell physiology, which is key to exploring novel therapeutic approaches in VRAC-associated pathologies.bestrophin 1 | volume-regulated anion channel | induced pluripotent stem cell | retinal pigment epithelium | mouse sperm

The extracellular matrix (ECM) of the brain plays crucial roles during the development, maturation, and regeneration of the CNS. In a subpopulation of neurons, the ECM condenses to superstructures called perineuronal nets (PNNs) that surround synapses. Camillo Golgi described PNNs a century ago, yet their biological functions remain elusive. Here, we studied a mouse mutant that lacks four ECM components highly enriched in the developing brain: the glycoproteins tenascin-C and tenascin-R and the chondroitin sulfate proteoglycans brevican and neurocan. Primary embryonic hippocampal neurons and astrocytes were cultivated using a cell insert system that allows for co-culture of distinct cell populations in the absence of direct membrane contacts. The wild-type and knock-out cells were combined in the four possible permutations. Using this approach, neurons cultivated in the presence of mutant astrocytes displayed a transient increase of synapses after 2 weeks. However, after a period of 3 weeks or longer, synapse formation and stabilization were compromised when either neuron or astrocyte cell populations or both were of mutant origin. The development of PNN structures was observed, but their size was substantially reduced on knock-out neurons. The synaptic activity of both wild-type and knock-out neurons was monitored using whole-cell patch clamping. The salient observation was a reduced frequency of IPSCs and EPSCs, whereas the amplitudes were not modified. Remarkably, the knock-out neuron phenotypes could not be rescued by wild-type astrocytes. We conclude that the elimination of four ECM genes compromises neuronal function.

No abstract

Phosphatidylinositol 3-kinase (PI3K)-dependent phosphoinositide signaling has been implicated in diverse cellular systems coupled to receptors for many different ligands, but the extent to which it functions in sensory transduction is yet to be determined. We now report that blocking PI3K activity increases odorant-evoked, cyclic nucleotide-dependent elevation of [Ca(2+)](i) in acutely dissociated rat olfactory receptor neurons and does so in an odorant-specific manner. These findings imply that 3-phosphoinositide signaling acts in vertebrate olfactory transduction to inhibit cyclic nucleotide-dependent excitation of the cells and that the interaction of the two signaling pathways is important in odorant coding, indicating that 3-phosphoinositide signaling can play a role in sensory transduction.