Olfactory transduction is thought to be mediated by a membrane-bound receptor protein initiating a multistep reaction cascade which ultimately leads to a depolarizing generator current. There is considerable evidence for the involvement of adenylate cyclase in vertebrate olfactory transduction, and some data indicate that phospholipase C may have a central role in insect olfaction. However, one must show that odorants not only stimulate enzyme activity but also induce changes in concentrations of relevant second messengers. One important criterion for a candidate second messenger of chemo-electrical transduction is that its formation must precede the onset of the odorant-induced membrane permeability changes which proceed on a subsecond time-scale. Here we report an odorant-induced, transient accumulation of cyclic AMP in isolated olfactory cilia from rats, and the generation of inositol trisphosphate in antennal preparations from insects, both of which show subsecond time courses that are sufficiently rapid to mediate the odorant-regulated permeability of olfactory receptor cells.

The global high prevalence of hypertension and cardiovascular disease has raised concerns regarding the sodium content of the foods which we consume. Over 75% of sodium intake in industrialized diets is likely to come from processed and restaurant foods. Therefore international authorities, such as the World Health Organisation, are encouraging the food industry to reduce sodium levels in their products. Significant sodium reduction is not without complications as salt plays an important role in taste, and in some products is needed also for preservation and processing. The most promising sodium reduction strategy is to adapt the preference of consumers for saltiness by reducing sodium in products in small steps. However, this is a time-consuming approach that needs to be applied industry-wide in order to be effective. Therefore the food industry is also investigating solutions that will maintain the same perceived salt intensity at lower sodium levels. Each of these has specific advantages, disadvantages, and time lines for implementation. Currently applied approaches are resulting in sodium reduction between 20-30%. Further reduction will require new technologies. Research into the physiology of taste perception and salt receptors is an emerging area of science that is needed in order to achieve larger sodium reductions.

Two closely related ␤ subunit mRNAs (xo28 and xo32) were identified in Xenopus oocytes by molecular cloning. One or both appear to be expressed as active proteins, because: (i) injection of Xenopus ␤ antisense oligonucleotides, but not of sense or unrelated oligonucleotides, significantly reduced endogenous oocyte voltage-gated Ca 2؉ channel (VGCC) currents and obliterated VGCC currents that arise after injection of mammalian ␣ 1 cRNAs (␣ 1C and ␣ 1E ); (ii) coinjection of a Xenopus ␤ antisense oligonucleotide and excess rat ␤ cRNA rescued expression of ␣ 1 Ca 2؉ channel currents; and (iii) coinjection of mammalian ␣ 1 cRNA with cRNA encoding either of the two Xenopus ␤ subunits facilitated both activation and inactivation of Ca 2؉ channel currents by voltage, as happens with most mammalian ␤ subunits. The Xenopus ␤ subunit cDNAs (␤3xo cDNAs) predict proteins of 484 aa that differ in only 22 aa and resemble most closely the sequence of the mammalian type 3 ␤ subunit. We propose that ''␣ 1 alone'' channels are in fact tightly associated ␣ 1 ␤3xo channels, and that effects of exogenous ␤ subunits are due to formation of higher-order [␣ 1 ␤]␤ n complexes with an unknown contribution of ␤3xo. It is thus possible that functional mammalian VGCCs, rather than having subunit composition ␣ 1 ␤, are [␣ 1 ␤]␤ n complexes that associate with ␣ 2 ␦ and, as appropriate, other tissue-specific accessory proteins. In support of this hypothesis, we discovered that the last 277-aa of ␣ 1E have a ␤ subunit binding domain. This ␤ binding domain is distinct from the previously known interaction domain located between repeats I and II of calcium channel ␣ 1 subunits.Xenopus oocytes translate exogenously injected mRNAs and cRNAs with relatively high efficiency. This has made them systems of choice for the functional expression and characterization of many cloned molecules, such as neuronal ligandgated ion channels, G protein-coupled receptors, and many voltage-gated ion channels, including voltage-dependent Ca 2ϩ channels. Voltage-dependent Ca 2ϩ channels are formed of an ␣ 1 pore-forming and voltage-sensing subunit and ␤ and ␣ 2 ␦ regulatory subunits. Functional expression in Xenopus oocytes has not only been used to define structure-function relations of voltage-gated calcium channels by assessing the effects of specific mutations of the ␣ 1 channel protein, but also to define identity and roles of the regulatory subunits in promoting ␣ 1 expression or modifying the properties of the expressed ␣ 1 subunit. Several nonallelic genes encoding ␣ 1 subunits, termed ␣ 1S and ␣ 1A -␣ 1E , have been identified by molecular cloning (1-4). Of these, all except ␣ 1S have been functionally expressed in the Xenopus oocyte. However, while certain variants of ␣ 1C and ␣ 1E can be expressed without coinjection of other subunit cRNAs (e.g., refs. 5 and 6), others, particularly ␣ 1A , yield only minimal currents in the absence of additional subunits, notably a ␤ subunit (7,8). The reasons for these differences are not understood.The interpreta...

The molecular mechanisms mediating the chemoelectrical signal transduction in olfactory receptor cells are still elusive. In this study odor induced formation of second messengers in rat olfactory cilia was monitored in a subsecond time range using a rapid kinetic device. Application of micromolar concentration of citralva induced a rapid, transient elevation of the cyclic adenosine monophosphate level, whereas the concentration of inositol trisphosphate was not affected. In contrast, pyrazine caused a rise in the concentration of inositol trisphosphate, not affecting the level of cyclic adenosine monophosphate. Analysis of the kinetic parameter for the odorant induced reaction indicated that apparently two systems are operating simultaneously.The activating effects of odorants appear to be mediated via different G-proteins. Thus, at least two different second messenger pathways appear to be involved in olfactory signal transduction.

Calcium channel beta subunits have profound effects on how alpha1 subunits perform. In this article we summarize our present knowledge of the primary structures of beta subunits as deduced from cDNAs and illustrate their different properties. Upon co-expression with alpha1 subunits, the effects of beta subunits vary somewhat between L-type and non-L-type channels mostly because the two types of channels have different responses to voltage which are affected by beta subunits, such as long-lasting prepulse facilitation of alpha1C (absent in alpha1E) and inhibition by G protein betagamma dimer of alpha1E, absent in alpha1C. One beta subunit, a brain beta2a splice variant that is palmitoylated, has several effects not seen with any of the others, and these are due to palmitoylation. We also illustrate the finding that functional expression of alpha1 in oocytes requires a beta subunit even if the final channel shows no evidence for its presence. We propose two structural models for Ca2+ channels to account for "alpha1 alone" channels seen in cells with limited beta subunit expression. In one model, beta dissociates from the mature alpha1 after proper folding and membrane insertion. Regulated channels seen upon co-expression of high levels of beta would then have subunit composition alpha1beta. In the other model, the "chaperoning" beta remains associated with the mature channel and "alpha1 alone" channels would in fact be alpha1beta channels. Upon co-expression of high levels of beta the regulated channels would have composition [alpha1beta]beta.

Laser scanning confocal microscopy in combination with the fluorescent calcium indicators Fluo-3 and Fura-Red was employed to estimate the intracellular concentration of free calcium ions in individual olfactory receptor neurons and to monitor temporal and spatial changes in the Ca(2+)-level upon stimulation. The chemosensory cells responded to odorants with a significant increase in the calcium concentration, preferentially in the dendritic knob. Applying various stimulation paradigma, it was found that in a population of isolated cells, subsets of receptor neurons display distinct patterns of responsiveness.

G-protein-mediated signalling processes are involved in sweet and bitter taste transduction. In particular, the G protein alpha-subunit gustducin has been implicated in these processes. One of the limiting factors for the time-course of cellular responses induced by tastants is therefore the intrinsic GTPase activity of alpha-gustducin, which determines the lifetime of the active G protein complex. In several signalling systems specific 'regulator of G protein signalling' (RGS) proteins accelerate the GTPase activity of G protein alpha-subunits. Using differential screening approaches, we have identified a novel RGS protein termed RGS21, which represents the smallest known member of this protein family. Reverse transcription polymerase chain reaction and in situ hybridization experiments demonstrated that RGS21 is expressed selectively in taste tissue where it is found in a subpopulation of sensory cells. Furthermore, it is coexpressed in individual taste cells with bitter and sweet transduction components including alpha-gustducin, phospholipase Cbeta2, T1R2/T1R3 sweet taste receptors and T2R bitter taste receptors. In vitro binding assays demonstrate that RGS21 binds alpha-gustducin in a conformation-dependent manner and has the potential to interact with the same Galpha subtypes as T1R receptors. These results suggest that RGS21 could play a regulatory role in bitter as well as sweet taste transduction processes.

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