Sweet-tasting substances can be grouped into four classes: carbohydrates (glucose, fructose, sucrose, lactose ...), amino acids (glycine), peptides (aspartame) and aliphatic alcohols (xylitol and saccharin). These substances are perceived sweet as they have the ability to bind receptor proteins that mediate the perception of sweet taste.
The aspartame has a sweetening power 200 times greater than sucrose: it means that at the same level of sweetness, the amount of aspartame used is significantly lower than the amount of sucrose that should be employed.
In fact sweetening power is a parameter, derived from the relationship between the concentration of a sucrose solution and the concentration of a solution of a sweetener with equal density of sweetness.
The transduction process is mediated by a family of C G protein-coupled receptors (GPCRs), T1Rs, which is selectively expressed in the taste buds. Hong Xu et al. in 2004 led a sophisticated study about sweet receptors, in which they discovered why these receptors are able to bind ligands with a very different structure as naturals and artificial sweeteners or inhibitors of sweet sensation like lactisole. On the other hand, receptors exhibits stereo-selectivity for certain molecules: for examples, it responds to D-thryptophan but not L-tryptophan. (Xu, H. et al. Different functional roles of T1R subunits in the heteromeric taste receptors, 2004)
Sweet receptor is a heterodimer formed by two seven-pass proteins T1R2 and T1R3 and both components are required to manifest a functional receptor. Recently it was shown that the gene sac, isolated in rats and determining the carriers' preference for saccharin, sucrose and other sweeteners, in mammals encodes the G-protein coupled receptor T1R3. (Nelson, G. et al. Mammalian Sweet Taste Receptors, 2001).
Consistent with the sensory and behavioral data (Xiadong Li et al.), human but not rat T1R2/T1R3 selectively responds to a group of sweeteners, including aspartame, neotame and cyclamate. Xu et al. used differences in agonist specificity by generating chimieric T1Rs between human an rat genes, with the junction located immediately before the transmembrane domain, in order to map their binding sites on the receptors. They transfected HEK-293 cells (Human Embryonic Kidney cells) with Galpha15/i1 (a chimeric Galpha15 protein with C-terminal tail sequence from Galphai1) and different combinations of the chimeric sweet receptors and tested their responses to aspartame, neotame and cycamate.
In figure B is explained how combination of T1R2R-H (N-term taken from rat receptor sequence and C-terminal tail from human one) and human T1R3 abolishes responses to aspartame and neotame. In order to detect receptors activity, researchers performed assays on HEK-293 for intracellular calcium increases in response to sweeteners.
Conversely, combination of T1R2H-R (human N-terminal and rat C-terminal) with rodent T1R3 doesn't affect responses to aspartame and neotame. This finding suggests N-terminal domain of human T1R2 is required for recognizing both aspartame and neotame.
In a second step, in order to narrow down the essential N-terminal aminoacids involved in recognition of these artificial sweeteners, they conduced mutagenesis studies on T1R2. By aligning rat and human T1R2 sequences, they selected three conserved amino acids on which they could act. The substitution of a tyrosine (Y218) abolished the responses to all sweeteners, suggesting that it might be important for the overall conformation of the sweet taste receptor. Instead, the substitution of a serine (S144) and a glutamic acid (E302) with alanine selectively affected the response to aspartame and neotame.
These data clearly indicate the existence of multiple binding pockets on the receptor for different classes of agonists, and that provides a possible explanation for the structural diversity of sweeteners.
Furthermore, T1R2 is also required to bind G protein. Xu et al. demonstrated that replacement of C-terminus of human T1R2 with the corresponding rat sequence abolished coupling. Gustducin has been proposed to be endogenous G protein for the sweet taste receptor.
The second component of heteromeric sweet receptor, T1R3, has a C-terminal transmembrane domain which is necessary to recognize the artificial sweetener sodium cyclamate. As shown in figure C, replacing T1R3 C-terminal domain extracellular loop 2 and 3 (EL2 and EL3) with rat sequences suppresses cyclamate response without affecting the sucrose or aspartame responses.
T1R3 also binds Lactisole, an aralkyl carboxylic acid, which acts as an inhibitors of this receptor by increasing detection thresholds for sweet. Usually it's widely used by food industries in order to mask exceeding sweet taste of candies. Since it is a non competitive inhibitor (inhibition does not depend on concentration of agonists and inhibitors) it doesn't bind the same cyclamate binding site but its own binding site on T1R3. Results shown in figure D demonstrate the importance of C-terminal domain in lactisole binding. HEK-293 cells with T1R3H-R (human N-terminal and rodant C-terminal) are not able to respond to lactisole: in fact perception of both sucrose and acesulfame K taste is not affected.
As already specified, the typical taste cells G protein is called gustducin. It allows to activate the cascade of events that leads to the perception of sweet. (SK McLaughlin et al. Gustducin is a taste-cell-specific G protein closely related to the transducins, 1992)
Transduction mechanisms activated by T1Rs are based on inositol triphosphate (IP3) pathway and cAMP pathway. This two pathways coexist in the same TCRs. In particular, according to Margolskee et al. (Margolskee RF et al. Molecular Mechanisms of Bitter and Sweet Taste Transduction,2002), all transduction pathways are proposed to converge on common elements that mediate a rise in intracellular Ca2+followed by neurotransmitter release. Artificial sweeteners activate GPCRs (T1R heterodimers) apparently linked via PLC to IP3 production and release of Ca2+ from intracellular stores. Sugars activate GPCRs (T1R heterodimers) apparently linked via adenylyl cyclase (AC) to cAMP production, which in turn may inhibit basolateral K+channels through phosphorylation by cAMP-activated protein kinase A (PKA).
In GPCR-Gq/Gbeta-gamma-IP3 pathway (Bernhardt SJ et al.), gustducin activates phospholipase PLCβ2 which hydrolyses the phosphatidyl-inositol-3,4-bisphosphate on the cell membrane to diacylglycerol and IP3. The increase in concentration of IP3 determines the release of calcium from internal stores. In turn, Ca2+ activates the ion channel TRMP5 that causes the influx of sodium and the depolarization of taste cells.
In GPCR-Gs-cAMP pathway (Stiem BJ et al.)(Naim M et al.)(Avenet P et al.), the gustducin activates adenyl cyclase which causes a consequent increase of cAMP.
This cyclic nucleotide may act directly to cause cation influx through cNMP-gated channels or indirectly activating protein kinase A (PKA). PKA phosphorylates and consequently closes K+-leak channels. The great amount of K+ trapped inside taste cell sets off a depolarization that opens calcium channels with the release of neurotransmitter.
Gustatory sensory cells are grouped in taste buds. Each bud contains different taste cells, each of them is specialized in the perception of a particular taste. For example, sweet taste is detected by type II sensitive cells; after depolarization, they release ATP through a mechanism that involves the transport across hemichannels made of a protein similar to the connexin, called pannexin 1. (Huang YJ et al.The role of pannexin 1 hemichannels in ATP release and cell-cell communication in mouse taste buds, 2007).
ATP, released as a neurotransmitter by gustatory cells, acts on inotropic receptors P2X2 and P2X3 present on afferent neurons, determining the direct excitation of the central taste way.
