Pergamon Camp. Biochem. Physiol. Vol. i09A, No. 2, pp. 361-367, 1994

Copyright 0 1994 Elsevier Science Ltd Printed in Great Britain. All rights reserved

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~~ectrophysio~ogi~a~ responses of ~~~~~~~ oocytes to amino acids: criteria for expression of injected mRNA coding chemoreceptors

Masaya Etoh and Kiyonori Yoshii Dept. of Biochemical Engineering and Science, Kyushu Institute of Technology, Iizuka, Fukuoka 820, Japan

Responses of endogenous transporters/receptors of Xenopus oocytes to L-alanine, L-arginine, L-leucine and L-serine were investigated under voltage clamp conditions. (a) Concen~atio~ response relations for the amino acids followed Langmuir’s adsorption isotherm. (b) The neutral amino acids required Na+ to elicit the responses, whereas L-arginine did not. (c) The responses to L-ala&e decreased with decreasing pH and became undetectablct at pH 5.5. The present experiments supply criteria to judge if the oocytes translate exogenous mRNA coding taste or olfactory receptor proteins for the amino acids, the best characterized stimuli, especially in fishes.

Key words: Voltage-clamp; Amino acids; Xenopus oocytes; Na +-dependence; pH- dependence; Kinetics; Current-voltage relations; Taste; Olfaction; mRNA.

Camp. ~ioc~em. P~ysi~l~ 109A, 361-367, 1994.

Xenopus oocytes are spherical, N 1 mm in diameter, and produce functional receptors or ion channels when they are injected with mRNA obtained from various sources (Gurdon et al., 1971). Therefore, the oocytes have been used as an appropriate expression tool for voltage clamp exper- iments (Numa, 1989; Sumikawa et al., 1986).

Several investigators (Dahmen et al., 1992; Getchell et ai., 1990; Yoshii and Kurihara, 1989) took advantage of this expression system to examine the olfactory and taste receptor mechanisms, especially those for amino acids. However, the oocytes, like other animal cells, have trans-

~~rre~po~~e~ce to: M. Etoh, Dept. of Biochemical Engineering and Science, Kyushu Institute of Technology, Iizuka, Fukuoka 820, Japan.

Received 16 September 1993; accepted 31 March 1994.

port systems for amino acids (Aoshima et al., 1988; Campa and Kilberg, 1989; Jung et al., 1984; Taylor et al., 1989; Van Winkle, 1988) and some of them are electrogenic. This could confound the analyses of ex- pressed chemoreceptor proteins. Also, in these investigations, no intensive measure- ment was carried out to clarify the degree of individual difference in the electrical re- sponses to amino acids, which becomes a critical factor in the expression experiments. In the present experiments, we investigated the electrophysiolo~cal responses of the endogenous transporters/receptors of Xenopus oocytes to provide criteria to sep- arate them from those examined by exogen- ous mRNA in injected oocytes.

Materials and Methods

Preparation of Xenopus oocytes

Stage V or VI oocytes were obtained as previously described (Yoshii et al., 1987).

361

362 M. Etoh and K. Yoshii

The oocytes were manually denuded and then incubated in Barth’s medium for l-3 hr before use.

Voltage clamp experiments

The oocytes were investigated under voltage clamp conditions using two glass electrodes filled with 3 M KC1 as previously reported (Yoshii et al., 1987). The com- mand voltage pulses were generated by a computer with a D/A converter and voltage-clamp currents were recorded by the computer with an A/D converter or a pen recorder. The experiments were carried out at room temperature.

Compositions of solutions

An oocyte in a recording chamber was continuously superfused with a physiologi-

L-leucine

cal solution (96 mM NaCl, 2 mM KCl, 2 mM CaCl,, 1 mM MgCl*, 10 mM HEPES (N-2-hydroxyethylpiperazine-N’- 2-ethanesulfonic acid), pH 7.4) referred to as a standard solution. Perfusing solutions of different pHs were prepared by substitut- ing HEPES with different buffers; MES (Z(N-morpholino) ethanesulfonic acid), Bis-Tris (2,2-bis(hydrocymethyl)-2,2’,2”- nitrilotriethanol), sodium acetate, or CHES (cyclohexylaminoethanesulfonic acid). A Na+ free solution was prepared by re- placing NaCl in the standard solution with choline Cl. Stimulating amino acid solutions were prepared by dissolving the amino acids in the appropriate perfusing solutions. Changes in pH of the solutions resulting from dissolving amino acids were compensated with NaOH or HCl.

3 x lo-5 M 3 x 10-4 M 3 x 10-3 M lo-2 M

=-Ti-$q+

L-alanine

10-S M 10-4 M 10-3 M 10-2 M I

--* =-G- -

L-arginine

10-6 M ‘02 M 10-4 M

L-serine 3 x 10-5 M 10-4 M 3 X 10-4 M 3 x 10-3 M 10-2 M

=ww =+wI;;nhm(+__ -

7r-

ml

Fig. 1. Voltage clamp currents as responses to different concentrations of L-leucine, L-alanine, L-arginine, and L-serine at holding potentials of - 50 mV. Arrow head shows off response. Downward deflections indicate inward currents. Numerals indicate concentrations of the amino acids. Horizontal bars on each trace; durations of stimulations. Vertical scale; 4 nA for

L-leucine, L-alanine, L-arginine, 2 nA for L-set-me. Horizontal scale: 1 min.

Responses of Xenopus oocytes to amino acids 363

10

1

Fig. 2. Distribution of responses recorded from 69 oocytes with holding potential of - 50 mV to 10 mM L-alanine. Inward currents larger than 1 nA are taken

as responses.

Chemicals used were of analytical grade and dissolved in deionized water prepared with Milli-Q SP (Millipore).

Stimulation

Stimulating solutions flowed into the recording chamber through a plastic tube (O-2 mm) positioned with a micromanipu- lator. For stimulation, the plastic tube was manually moved near the oocyte. Care was taken to minimize mechanical stimulation, which was especially important in alkaline solutions since the oocytes became more sensitive under these conditions.

Eflects of changes in osmolarity

Since the osmolarity of the stimulating solutions was different from the perfusing solutions, we examined the effect of changes in osmolarity using 20 mM sucrose dis- solved in the standard solution. The re- sponses elicited by the sucrose solution were less than 1 nA, indicating that the stimulating effect was negligible. In the present experiments, the voltage-clamp cur- rents larger than 1 nA were used as the responses.

Results Voltage -clamp currents

Figure 1 shows representative responses to various amino acids of the oocytes voltage-clamped at -50 mV. All amino acids elicited inward currents at the concen- trations tested. L-Leucine elicited off re- sponses (the responses that appear when stimulating solutions are washed away) at higher concentrations, typically at 10m2 M (arrow head). L-Alanine, L-arginine and L-sex-me did not produce clear off responses, even at high concentrations.

Most of the oocytes responded to the amino acids, and there was no clear re- lationship between the magnitude of re- sponses and the mothers supplying the oocytes. As shown in Fig. 2, 65 out of 69 oocytes responded to L-alanine. The re- sponses of most were less than 20 nA, although a few cells produced a response between 20 and 37.8 nA. Table 1 summar- izes the means and standard deviations of the responses to various amino acids. The mean of the responses to L-alanine is 9.8 nA. The order of the inward currents is L-alanine > L-arginine > L-leucine > L-ser- ine, although there is no significant differ- ence between the magnitude of the re- sponses to L-leucine and L-arginine. Also, the confidence interval for population means of these amino acids did not involve zero (P < 0.05).

Concentration-response curves

Figure 3 plots the representative re- sponses to various amino acids as a logar- ithmic function of amino acid concentrations, where the oocyte was voltage-clamped at - 50 mV. The responses to L-leucine appeared at lo-’ M, increased with increasing concentrations and satu- rated at 10e2 M (Fig. 3A). Solid lines are least-squares fits of the Hill equation to

Table 1. Responses of oocytes to 10 mM amino acids

Ala Leu Arg Ser

Responses (nA) 9.8 & 6.7 5.1 f 3.9 5.9 * 2.8 2.5 +_ 1.7 Number of oocytes 65 94 36 70

Means and standard deviations are represented. Holding potential; - 50 mV. t-test revealed significant inter-amino acids differences (P < 0.01) except between L-leucine and L-arginine. Ala, Leu, Arg, and Ser are -abbreviations of L-alanine, L-leucine, L-arginine, and L-serine, respectively.

364 M. Etoh and K. Yoshii

08 I lo" lU5 10'4 m3 10'2

[ L-leucine 1 (M) D4 r

[ L-arginine 1 (M)

Yll-LL ‘:- ’ .A! ’ 10-a 10-5 lo4 lo3 lo* 10-E 10-s 10' 10-3 lO-*

[ L-aianine ] (M) [L-swine] (M)

Fig. 3. Responses to various concentrations of L-leucine (A), L-alanine (B), L-arginine (C), and L-serine (D) as a function of concentrations of the amino acids. Each curve is obtained from

different oocytes.

the data with parameters, R,,, (maximal response), I& (apparent dissociation constant), and Hill’s n (Hill’s coefficient). As the figure shows, the concen- tration-response relations follow the equation.

The responses to L-alanine (Fig. 3B), L-arginine (Fig. 3C), and L-serine (Fig. 3D) also follow the equation although, except for the responses to L-arginine, they do not saturate up to lop2 M. The threshold con- centrations are L-alanine, 10m5 M; L-serine, 10m4 M; L-arginine, lop6 M.

Table 2 summarizes means and standard deviations of parameters used in the least- squares fits. The parameters for L-alanine and L-serine may be unreliable because they do not reach the respective saturation

levels. The order of maximal responses is L- alanine > L-leucine > L-arginine > L-serine. The order of apparent dissociation con- stants is L-arginine < L-leucine < L-ser- ine < L-alanine. Hill’s coefficients are close to 1.0, indicating that Langmuir’s adsorp- tion isotherm describes the concen- tration-response relations.

Current-voltage relations

The current-voltage relationships for the amino acid responses were investigated by voltage-clamping the membrane potentials in a stepwise manner. The current-voltage relations were almost linear, but the rever- sal potentials were different from oocyte to oocyte.

Table 2. Parameters used in least squares fits

Ala Leu Arg Ser

R,,, (nA) 10 f 5.1 7.1 +_ 2.5 5.9 + 0.96 &(mM) 5.1 + 4.3 0.41 f 0.35 0.15 * 0.03 ::; Hill’s II 0.91 f 0.11 0.99 * 0.02 0.99 + 0.02 1.0 Number of oocvtes 4 3 3 2

Means and standard deviations are represented. For L-serine, means only. t-test revealed no significant inter-amino acids differences among Rmaxs, Kds, and Hill’s n (P < 0.01).

Responses of Xenopus oocytes to amino acids 365

nA 10

1 10-2 M

L-alanlne 10-Z RR

L-arginlne

I B +Na+

V

Ii- If-

Fig. 4. Current-voltage relations for L-leucine (o), L-alanine (O), and t-arginine (a) recorded from the

same oocyte.

Figure 4 shows current-voltage relations obtained from the same oocyte for different amino acids. The I-V relation for L-leucine is nearly linear at negative membrane po- tentials, but becomes non-linear positive to 10 mV. L-Alanine yields a similar cur- rent-voltage curve. The reversal potentials for these amino acids are similar; L-leucine, 33 mV; L-alanine, 35 mV.

In contrast to the neutral amino acids, L-arginine yields a linear current-voltage relation over the entire range of membrane potentials. The currents do not reverse di- rection within the range of the membrane potentials tested (-90 to +48 mV).

Although similar results were obtained from many oocytes, reversal potentials were not observed in three oocytes out of eight for L-alanine and one out of four for L- leucine. Also, in two oocytes out of 11, reversal potentials (25 and 32 mV) were recorded for r,-arginine.

Table 3 summarizes the means and stan- dard deviations of slope conductance and reversal potentials, respectively. The order of slope conductance is L-alanine > L- leucine > L-arginine. The mean reversal po- tentials for L-alanine and L-leucine do not differ significantly.

Fig. 5. Na + dependence of responses to L-alanine and L-arginine. +Na+ and -Na+ indicate the responses recorded in the standard and Na+ free solution,

respectively.

Na + ~pe~de~ce

The Na+ dependence of the responses to the amino acids was investigated by replac- ing Na+ in the bathing solution with choline. As Fig. 5 shows, the response to L-alanine was decreased by removal of Na+, whereas the response to L-arginine was practically unchanged. t-Leucine and L-serine also required Na+ to elicit the responses (data not shown).

pH dependence

The responses to t-alanine depended on the pH of the stimulating solutions. As Fig. 6 shows, 10 mM L-alanine at pH 9.0 elicited a large response. The response decreased with decreasing pH and became undetect- able at pH -5. Although the responses depended on the pH of the solutions, they were independent of the species of the buffers ZNa acetate (pH 5.2), Bis-Tris (PH 5.5) or MES (pH 5.0)]. This indicates that the suppression resulted from lowering pH rather than binding of the buffers to the transporters.

Table 3. Slope conductance (nS) for 1OmM amino acids

Ala Leu Arg

Slope conductance 1525 119(8) 77 + 33 (4) 52 + 21(10) Reversal potentials 29 & 12 (5) 38 + 5 (3)

Means and standard deviations are represented. Numerals in parenthesis are number of oocytes. t-test revealed no significant inter-amino acids differences in slope ~nductan~ (P < 0.02, except between L-afanine and L-arginine) and in reversal potentials (P < 0.01).

366 M. Etoh am d K. Yoshii

2.0 r f

,g 2 1.0 - /

q

t ‘i; 2 Jo t

0.0 /(,

-4 I b I

4 5 7 0 8 5 10

PH

Fig. 6. Relative magnitude of responses to 1OmM L-alanine as a function of pH of the stimulating solutions where the magnitude of the responses at pH 7.4 is taken as unity. Line is drawn by eye. A, MES; n , CH3COOH; 0, Bis-Tris; 0, HEPES; 0, CHES.

Discussion

The present experiments revealed that several amino acids elicited substantial re- sponses from Xenopus oocytes. These re- sponses may interfere with the investigation of receptor mechanisms of exogenous amino acid chemoreceptors produced by Xenopus oocytes. However, the magnitude of the responses, concentration-response relations, and Na+ and pH-dependence characterized in the present experiments should provide criteria to determine if an oocyte expresses exogenous mRNA.

For example, although carp olfactory receptor cells (Yoshii and Kurihara, 1983b) and eel gustatory receptor cells (Yoshi and Kurihara, 1983a) required cations to elicit neural responses to amino acids, any cat- ions including K + , Mg*+ , or organic cations supported the responses. Thus, when the responses of mRNA-injected oocytes devi- ate significantly from those presented here, it is likely that the oocytes have translated functional receptors.

The responses to the neutral amino acids were Na+-dependent and their reversal po- tentials of - 35 mV were close to the equi- librium potential of Na+, between 37 and 117 mV (Baud, 1983; Baud et al., 1982; Kusano et al., 1982). Therefore, it is likely that Na +-dependent systems mediate a significant component of the responses to neutral amino acids.

The reversal potentials for the amino acids were different from oocyte to oocyte. Although the reversal potentials for the neutral amino acids were between 0 and 50 mV in most oocytes, they were greater

than 50 mV in some cells. Also, although the r.-arginine response usually did not re- verse, a few oocytes displayed a reversal of about - 30 mV. There was no obvious re- lation between reversal potentials and the female donor. These results suggest that each Xenopus oocyte has several receptor/ transporter systems, which are present in different ratios.

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