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.2012 Feb 28;109(9):3492-7.
doi: 10.1073/pnas.1111297109. Epub 2012 Feb 10.

Crucial role of copper in detection of metal-coordinating odorants

Affiliations

Crucial role of copper in detection of metal-coordinating odorants

Xufang Duan et al. Proc Natl Acad Sci U S A..

Abstract

Odorant receptors (ORs) in olfactory sensory neurons (OSNs) mediate detection of volatile odorants. Divalent sulfur compounds, such as thiols and thioethers, are extremely potent odorants. We identify a mouse OR, MOR244-3, robustly responding to (methylthio)methanethiol (MeSCH(2)SH; MTMT) in heterologous cells. Found specifically in male mouse urine, strong-smelling MTMT [human threshold 100 parts per billion (ppb)] is a semiochemical that attracts female mice. Nonadjacent thiol and thioether groups in MTMT suggest involvement of a chelated metal complex in MOR244-3 activation. Metal ion involvement in thiol-OR interactions was previously proposed, but whether these ions change thiol-mediated OR activation remained unknown. We show that copper ion among all metal ions tested is required for robust activation of MOR244-3 toward ppb levels of MTMT, structurally related sulfur compounds, and other metal-coordinating odorants (e.g., strong-smelling trans-cyclooctene) among >125 compounds tested. Copper chelator (tetraethylenepentamine, TEPA) addition abolishes the response of MOR244-3 to MTMT. Histidine 105, located in the third transmembrane domain near the extracellular side, is proposed to serve as a copper-coordinating residue mediating interaction with the MTMT-copper complex. Electrophysiological recordings of the OSNs in the septal organ, abundantly expressing MOR244-3, revealed neurons responding to MTMT. Addition of copper ion and chelator TEPA respectively enhanced and reduced the response of some MTMT-responding neurons, demonstrating the physiological relevance of copper ion in olfaction. In a behavioral context, an olfactory discrimination assay showed that mice injected with TEPA failed to discriminate MTMT. This report establishes the role of metal ions in mammalian odor detection by ORs.

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Conflict of interest statement

Conflict of interest statement: The authors declare a potential conflict of interest. The authors plan to submit a patent application relevant to the work.

Figures

Fig. 1.
Fig. 1.
MOR244-3 is selectively activated by MTMT and other sulfur-containing odors. Responses of MOR244-3 to (A) MTMT and a sulfurous odor panel containing 19 MTMT analogs and 24 other sulfur-containing compounds and (B) a diverse odor panel containing 89 compounds. The concentration of all odors used was 30 μM, except when cell toxicity was observed, the maximally tolerant concentrations were used, including 0.3 μM for 2-(methylthio)ethanethiol and 10 μM for 2-mercaptobenzothiazole and 2-mercaptopyridine. All responses are normalized to the response to MTMT. For all figures, normalized luciferase responses are shown as mean ± SEM (n = 3). Odorants are color-coded by structural features and functional groups. For odorants with multiple functional groups, the functional group with the highest oxidation state is used for categorization.
Fig. 2.
Fig. 2.
Copper ion enhances the activation of MOR244-3 in response to MTMT. (A) Dose–response curves of MOR244-3 against 30 μM MTMT with increasing concentrations of various metal ions supplemented in the cell culture medium. (B) Dose–response curves of MOR244-3 against increasing concentrations of MTMT with the concentration of different metals held constant at 30 μM, except for PtCl2 and AgNO3, which were 10 μM and 5 μM, respectively. (C) Real-time measurement of MOR244-3 activation in the presence of 30 μM Cu2+ and/or 30 μM MTMT as measured by a GloSensor assay within 30 min of odorant addition. Arrow indicates odorant addition. (D) Responses of MOR244-3 to 0 and 30 μM MTMT with 10 μM Cu2+ or TEPA and increasing the amount of TEPA or Cu2+, respectively. In this and subsequent figures, all responses are normalized to the highest response to MTMT. ForC,y axis represents luminescence, shown as mean ± SEM (n = 6).
Fig. 3.
Fig. 3.
Structural requirements of the copper ion enhancement effect of MOR244-3. (A) Structural relationship between MTMT and its analogs. Odors boxed with solid lines are those with prominent responses in the presence of 30 μM Cu2+, and odors boxed with dashed lines are those with less prominent responses, as defined by a more than 50% reduction in efficacy compared with MTMT. Unboxed odors did not elicit MOR244-3 response. (B) Dose–response curves of MOR244-3 to representative sulfur-containing compounds with and without 30 μM exogenous Cu2+addition. For odors with a significant response in the absence of exogenous Cu2+, as defined arbitrarily by a top value greater than 0.32, dose–response curves with 30 μM of TEPA are also shown. The chemical structures of the respective compounds are shown in the upper left-hand corners.F tests were used to compare the pairs of dose–response curves with or without Cu2+. Asterisks represent significance ofP values after Bonferonni corrections. **P < 0.01 (n = 3).
Fig. 4.
Fig. 4.
Physiological relevance of copper in the detection of MTMT. (A–D) MTMT responsive OSNs are present in the SO. (A) In a single non-SR1 cell, inward currents were elicited by MTMT pulses with varying length from 0.025 to 0.5 s. (B) The dose–response curve was fitted by grouping the data from six cells tested by MTMT pulses with different lengths. For each cell, the peak current induced by a pulse was normalized to the maximum response in that cell. (C) In some cells, the MTMT (10 μM) response was facilitated by adding 30 μM Cu2+. (D) In some cells, the MTMT response (10 μM) was reduced by adding 30 μM TEPA. InC andD, the thick trace was averaged from three trials under that condition (thin lines). All recordings were performed under voltage clamp mode with a holding potential of −60 mV. (E) Mice were trained to associate either eugenol or MTMT with sugar reward. On the test day, mice were injected with dH2O or TEPA and then tested for the ability to discriminate the two odors.Left: TEPA injection specifically abolishes olfactory detection of MTMT.Right: Recovery of olfactory discrimination ability for eugenol and MTMT 2 d after TEPA injection. Two days after the initial testing, mice were retested for the recovery of the ability to discriminate the two odors. They axis represents time spent investigating each odorant during the 9-min test period, shown as mean ± SEM. Pairedt test was used to compare the investigation times between groups. *P < 0.05 (n = 4). (F) A model for OR activation involving copper ion as a cofactor with the copper ion binding to the ligand for subsequent binding of the copper ion–odor complex to the OR.
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References

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