Early Proton Transfer Reaction in a Primate Blue-Sensitive Visual Pigment

The proton transfer reaction belongs to one of the key triggers for the functional expression of membrane proteins. Rod and cone opsins are light-sensitive G-protein-coupled receptors (GPCRs) that undergo the cis–trans isomerization of the retinal chromophore in response to light. The isomerization event initiates a conformational change in the opsin protein moiety, which propagates the downstream effector signaling. The final step of receptor activation is the deprotonation of the retinal Schiff base, a proton transfer reaction which has been believed to be identical among the cone opsins. Here, we report an unexpected proton transfer reaction occurring in the early photoreaction process of primate blue-sensitive pigment (MB). By using low-temperature UV–visible spectroscopy, we found that the Lumi intermediate of MB formed in transition from the BL intermediate shows an absorption maximum in the UV region, indicating the deprotonation of the retinal Schiff base. Comparison of the light-induced difference FTIR spectra of Batho, BL, and Lumi showed significant α-helical backbone C=O stretching and protonated carboxylate C=O stretching vibrations only in the Lumi intermediate. The transition from BL to Lumi thus involves dramatic changes in protein environment with a proton transfer reaction between the Schiff base and the counterion resulting in an absorption maximum in the UV region

Protein-Bound Water Molecules in Primate Red- and Green-Sensitive Visual Pigments

Protein-bound water molecules play crucial roles in the structure and function of proteins. The functional role of water molecules has been discussed for rhodopsin, the light sensor for twilight vision, on the basis of X-ray crystallography, Fourier transform infrared (FTIR) spectroscopy, and a radiolytic labeling method, but nothing is known about the protein-bound waters in our color visual pigments. Here we apply low-temperature FTIR spectroscopy to monkey red (MR)- and green (MG)-sensitive color pigments at 77 K and successfully identify water vibrations using D₂O and D₂¹⁸O in the whole midinfrared region. The observed water vibrations are 6–8 for MR and MG, indicating that several water molecules are present near the retinal chromophore and change their hydrogen bonds upon retinal photoisomerization. In this sense, color visual pigments possess protein-bound water molecules essentially similar to those of rhodopsin. The absence of strongly hydrogen-bonded water molecules (O–D stretch at <2400 cm^–1) is common between rhodopsin and color pigments, which greatly contrasts with the case of proton-pumping microbial rhodopsins. On the other hand, two important differences are observed in water signal between rhodopsin and color pigments. First, the water vibrations are identical between the 11-<i>cis</i> and 9-<i>cis</i> forms of rhodopsin, but different vibrational bands are observed at >2550 cm^–1 for both MR and MG. Second, strongly hydrogen-bonded water molecules (2303 cm^–1 for MR and 2308 cm^–1 for MG) are observed for the all-<i>trans</i> form after retinal photoisomerization, which is not the case for rhodopsin. These specific features of MR and MG can be explained by the presence of water molecules in the Cl^–-biding site, which are located near positions C11 and C9 of the retinal chromophore. The averaged frequencies of the observed water O–D stretching vibrations for MR and MG are lower as the λ_max is red-shifted, suggesting that water molecules are involved in the color tuning of our vision

RT-qPCR gustducin_English

RT-qPCR gustducin_Englis

RT-qPCR T1RsT2Rs_English

RT-qPCR T1RsT2Rs_Englis

RT-qPCR TRPM5_English

RT-qPCR TRPM5_Englis

Expression Analysis of Taste Signal Transduction Molecules in the Fungiform and Circumvallate Papillae of the Rhesus Macaque, <em>Macaca mulatta</em>

<div><p>The molecular mechanisms of the mammalian gustatory system have been examined in many studies using rodents as model organisms. In this study, we examined the mRNA expression of molecules involved in taste signal transduction in the fungiform papillae (FuP) and circumvallate papillae (CvP) of the rhesus macaque, <em>Macaca mulatta</em>, using <em>in situ</em> hybridization. <em>TAS1R1</em>, <em>TAS1R2, TAS2Rs, and PKD1L3</em> were exclusively expressed in different subsets of taste receptor cells (TRCs) in the FuP and CvP. This finding suggests that TRCs sensing different basic taste modalities are mutually segregated in macaque taste buds. Individual <em>TAS2Rs</em> exhibited a variety of expression patterns in terms of the apparent level of expression and the number of TRCs expressing these genes, as in the case of human <em>TAS2Rs</em>. <em>GNAT3</em>, but not <em>GNA14</em>, was expressed in TRCs of FuP, whereas <em>GNA14</em> was expressed in a small population of TRCs of CvP, which were distinct from <em>GNAT3</em>- or <em>TAS1R2</em>-positive TRCs. These results demonstrate similarities and differences between primates and rodents in the expression profiles of genes involved in taste signal transduction.</p> </div

The co-expression relationships among taste receptors and G protein α subunits.

<p>(A) In the CvP, <i>GNA14</i> was expressed in a much smaller population of TRCs than <i>GNAT3</i> and in a mutually exclusive manner. The <i>GNA14</i>–positive TRCs were distinct from those expressing <i>TAS1R2</i> and <i>TAS2R13</i>, but they were subsets of the <i>TAS1R3</i>-positive TRCs and partially overlapped with the <i>TAS1R1</i>-positive TRCs. n ≥1 (numbers of sections ≥2). (B) In the CvP, <i>TAS1R2</i> and <i>TAS2R13</i> were expressed in subsets of the <i>GNAT3</i>–positive TRCs, which partially overlapped with the <i>TAS1R1</i>- and <i>TAS1R3</i>-positive TRCs. n ≥2 (numbers of sections ≥4). (C) In the FuP, <i>TAS1R1</i>, <i>TAS1R2</i>, <i>TAS1R3</i>, and <i>TAS2R13</i> were expressed in subsets of <i>GNAT3</i>-positive TRCs. n ≥1 (numbers of sections ≥10). (D) Venn diagram illustrating the co-expression relationships among taste receptors and signal transduction molecules in the macaque and the mouse. Scale bars: 50 µm.</p

The co-expression relationships among taste receptors.

<p>(A) In the CvP, the <i>TAS1R3</i>-positive TRCs were negative for <i>TAS2R13</i>. The <i>PLCB2</i>-positive TRCs, which include <i>TAS1R1</i>-, <i>TAS1R2</i>-, <i>TAS1R3</i>-, and <i>TAS2R13</i>-positive TRCs, were negative for <i>PKD1L3</i>. n = 1 (numbers of sections ≥2). (B) In the FuP, the <i>TAS1R3</i>-positive TRCs were negative for <i>TAS2R13</i>. The <i>PLCB2</i>-positive TRCs, which include <i>TAS1R1</i>-, <i>TAS1R2</i>-, <i>TAS1R3</i>-, and <i>TAS2R13</i>-positive TRCs, were negative for <i>PKD1L3</i>. n = 1 or 2 (numbers of sections ≥10). Scale bars: 50 µm.</p

The mRNA expression of genes encoding taste receptors and signal transduction molecules in the fungiform and circumvallate papillae of the rhesus macaque.

<p>(A) <i>In situ</i> hybridization revealed that three <i>TAS1Rs</i>, <i>TAS2R13</i>, <i>PKD1L3</i>, <i>GNAT3</i>, <i>GNA14</i>, and <i>PLCB2</i> were robustly expressed in subsets of the TRCs in the CvP. These genes, except for <i>GNA14</i>, were also expressed in subsets of the TRCs in the FuP. n≥2 (numbers of sections ≥4) for <i>TAS1R1</i>, <i>TAS1R2</i>, <i>TAS1R3</i>, <i>PKD1L3</i>, <i>GNAT3</i>, and <i>TAS2R13</i> in CvP, n = 1 (numbers of sections ≥2) for <i>GNA14</i> and <i>PLCB2</i> in CvP, n≥2 (numbers of sections ≥20) for <i>TAS1R1</i>, <i>TAS1R2</i>, <i>TAS1R3</i>, <i>PKD1L3</i>, and <i>GNA14</i> in FuP, n = 1 (numbers of sections ≥10) for <i>GNAT3</i>, <i>PLCB2</i>, and <i>TAS2R13</i> in FuP. (B) The <i>TAS2Rs</i> located on chromosome 11 (<i>TAS2R9</i> and <i>TAS2R12-25</i>) appeared to be robustly expressed in subsets of TRCs, whereas only weak signals were observed for the <i>TAS2Rs</i> located on chromosomes 3 (<i>TAS2R1-8</i> and <i>TAS2R10-11</i>) and 6 (<i>TAS2R26</i>). <i>Tas2Rs</i> are arranged according to the locations on the chromosomes (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0045426#pone.0045426.s001" target="_blank">Figure S1</a>). n = 2 (numbers of sections ≥4) for <i>TAS2R1-8</i>, <i>10</i>-<i>11</i>, <i>21</i>, <i>23</i>, and <i>26</i>, n = 1 (numbers of sections ≥2) for <i>TAS2R9</i>, <i>12, 14</i>-<i>20</i>, <i>22</i>, and <i>24</i>-<i>25</i>. Scale bars: 50 µm.</p

The co-expression relationships among three <i>TAS1Rs</i>.

<p>(A) <i>TAS1R1</i> and <i>TAS1R2</i> were exclusively expressed in different subsets of the <i>TAS1R3</i>-positive TRCs in the CvP. <i>In situ</i> hybridization using a mixed probe for <i>TAS1R1</i> and <i>TAS1R2</i> combined with a probe for <i>TAS1R3</i> revealed the presence of TRCs expressing <i>TAS1R3</i> alone. n = 2 (numbers of sections ≥4). (B) <i>TAS1R1</i> and <i>TAS1R2</i> were exclusively expressed in different subsets of the <i>TAS1R3</i>-positive TRCs in the FuP. The <i>TAS1R3</i>-positive TRCs in the FuP and CvP were classified into three types of cells: cells expressing <i>TAS1R1</i>+<i>TAS1R3</i>, those expressing <i>TAS1R2</i>+<i>TAS1R3</i>, and those expressing <i>TAS1R3</i> alone. n = 1 or 2 (numbers of sections ≥10). Scale bars: 50 µm.</p