Odorant induced Cl− currents, resulting from cAMP mediated activation of the co-expressed CFTR reporter channel (Uezono et al., 1993 (link)), were measured 2-4 days after cRNA injection using two-electrode voltage clamp in an automated parallel electrophysiology system (OpusExpress 6000A, Molecular Devices). Micropipettes were filled with 3M KCl and had resistances of 0.2-2.0 MΩ. The holding potential was −70 mV. Current responses, filtered (4-pole, Bessel, low pass) at 20 Hz (−3db) and sampled at 100 Hz, were captured and stored using OpusXpress 1.1 software (Molecular Devices). Initial analysis was done using Clampfit 9.1 software (Molecular Devices). Oocytes were perfused with ND96 (in mM: 96 NaCl, 2 KCl, 1 CaCl2, 1 MgCl2, 5 HEPES, pH 7.5). Odorants were stored under argon and high concentration (1M) stock solutions of each odorant were prepared in DMSO. Each odorant, diluted in ND96, was applied for 15 sec (Abaffy et al., 2006 (link)). IBMX (1 mM) was used to activate the CFTR in a receptor independent manner. This occurs both through the inhibition of phosphodiesterase and consequent increase in cAMP concentration, and through a direct action on the CFTR (Schultz et al., 1999 (link)). The CFTR can be directly activated by a wide variety of structures (Ma et al., 2002 (link)). Thus, to guard against false positives, all compounds (at all concentrations) used in our studies were tested with oocytes expressing Gαolf and CFTR, but no odorant receptors (termed “R-” oocytes). Oocytes are generally stable under voltage clamp for one to several hours, providing a useful platform for ligand screening. The oocytes used to screen MOR23-1 (recordings from one of these oocytes are shown in Fig. 2 ) were unusually stable, providing 8 hours of recordings. All four recordings in Figure 2 were obtained from the same oocyte (one of 8 oocytes in the screen).
To explore the molecular receptive range, each MOR was screened with a panel of odorants, each at 100μM. The odorant panel consisted of 41 saturated, aliphatic primary alcohols, aldehydes, monocarboxylic acids, bromocarboxylic acids and dicarboxylic acids, ranging in length from 4 to 12 carbons. Some bromocarboxylic acids in the length series could not be obtained. Odorants were applied for 15 sec (Abaffy et al., 2006 (link)), followed by a 10 min wash with ND-96. Each odorant that elicited a statistically significant MOR response at 100 μM was also screened at 30 μM, 10 μM and 3 μM. The statistical significance of receptor responses was determined by comparison to the response of R- oocytes to the same odorant. For each odorant yielding a response, the mean R- response for that odorant was subtracted from the MOR response and the resulting value was then normalized to the response of the same oocyte to 100 μM of the normalizing ligand (nonanoic acid for MOR23-1, MOR32-11 and MOR40-4; undecanoic acid for MOR31-4). These normalized responses are presented as mean ± SEM. We previously observed that small to moderate current responses (≤ 1.5 μA) could be elicited repeatedly, with no loss of amplitude (Abaffy et al., 2006 (link)). However, when current amplitudes are large (> 1.5 μA), subsequent current responses can be partially suppressed for many minutes (data not shown). This suppression does not correlate with odorant concentration, but instead appears to be associated with large current amplitudes, suggesting that this temporary suppression occurs within the signal transduction pathway. Thus, during ligand screening, applications that occurred less than 30 minutes following a large response (> 1.5 μA) were redone with a separate set of oocytes. For dose-response analysis, each odorant response was normalized to an immediately preceding normalizing application (3 μM octanoic acid for MOR23-1; 3 μM dodecanoic acid for MOR31-4; 30 μM octanoic acid for MOR32-11; 30 μM undecanal for MOR40-4). Normalized data were fit using Prism 4 (Graphpad, San Diego, CA) according to the equation: I = Imax/(1+(EC50/X)n) where I represents the current response at a given concentration of odorant, X; Imax is the maximal response; EC50 is the concentration of odorant yielding a half maximal response; n is the apparent Hill coefficient. Statistical significance was assessed with Prism 4 (Graphpad, San Diego, CA) using a two-tailed unpaired t-test, or a one-way ANOVA followed by the Dunnett's post-test, as appropriate.
To explore the molecular receptive range, each MOR was screened with a panel of odorants, each at 100μM. The odorant panel consisted of 41 saturated, aliphatic primary alcohols, aldehydes, monocarboxylic acids, bromocarboxylic acids and dicarboxylic acids, ranging in length from 4 to 12 carbons. Some bromocarboxylic acids in the length series could not be obtained. Odorants were applied for 15 sec (Abaffy et al., 2006 (link)), followed by a 10 min wash with ND-96. Each odorant that elicited a statistically significant MOR response at 100 μM was also screened at 30 μM, 10 μM and 3 μM. The statistical significance of receptor responses was determined by comparison to the response of R- oocytes to the same odorant. For each odorant yielding a response, the mean R- response for that odorant was subtracted from the MOR response and the resulting value was then normalized to the response of the same oocyte to 100 μM of the normalizing ligand (nonanoic acid for MOR23-1, MOR32-11 and MOR40-4; undecanoic acid for MOR31-4). These normalized responses are presented as mean ± SEM. We previously observed that small to moderate current responses (≤ 1.5 μA) could be elicited repeatedly, with no loss of amplitude (Abaffy et al., 2006 (link)). However, when current amplitudes are large (> 1.5 μA), subsequent current responses can be partially suppressed for many minutes (data not shown). This suppression does not correlate with odorant concentration, but instead appears to be associated with large current amplitudes, suggesting that this temporary suppression occurs within the signal transduction pathway. Thus, during ligand screening, applications that occurred less than 30 minutes following a large response (> 1.5 μA) were redone with a separate set of oocytes. For dose-response analysis, each odorant response was normalized to an immediately preceding normalizing application (3 μM octanoic acid for MOR23-1; 3 μM dodecanoic acid for MOR31-4; 30 μM octanoic acid for MOR32-11; 30 μM undecanal for MOR40-4). Normalized data were fit using Prism 4 (Graphpad, San Diego, CA) according to the equation: I = Imax/(1+(EC50/X)n) where I represents the current response at a given concentration of odorant, X; Imax is the maximal response; EC50 is the concentration of odorant yielding a half maximal response; n is the apparent Hill coefficient. Statistical significance was assessed with Prism 4 (Graphpad, San Diego, CA) using a two-tailed unpaired t-test, or a one-way ANOVA followed by the Dunnett's post-test, as appropriate.
