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J Neurochem
2012 Dec 01;1236:911-8. doi: 10.1111/jnc.12013.
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Xenopus laevis RIC-3 enhances the functional expression of the C. elegans homomeric nicotinic receptor, ACR-16, in Xenopus oocytes.
Bennett HM
,
Lees K
,
Harper KM
,
Jones AK
,
Sattelle DB
,
Wonnacott S
,
Wolstenholme AJ
.
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RIC-3 enhances the functional expression of certain nicotinic acetylcholine receptors (nAChRs) in vertebrates and invertebrates and increases the availability of functional receptors in cultured cells and Xenopus laevis oocytes. Maximal activity of RIC-3 may be cell-type dependent, so neither mammalian nor invertebrate proteins is optimal in amphibian oocytes. We cloned the X. laevis ric-3 cDNA and tested the frog protein in oocyte expression studies. X. laevis RIC-3 shares 52% amino acid identity with human RIC-3 and only 17% with that of Caenorhabditis elegans. We used the C. elegans nicotinic receptor, ACR-16, to compare the ability of RIC-3 from three species to enhance receptor expression. In the absence of RIC-3, the proportion of oocytes expressing detectable nAChRs was greatly reduced. Varying the ratio of acr-16 to X. laevis ric-3 cRNAs injected into oocytes had little impact on the total cell current. When X. laevis, human or C. elegans ric-3 cRNAs were co-injected with acr-16 cRNA (1 : 1 ratio), 100 μM acetylcholine induced larger currents in oocytes expressing X. laevis RIC-3 compared with its orthologues. This provides further evidence for a species-specific component of RIC-3 activity, and suggests that X. laevis RIC-3 is useful for enhancing the expression of invertebrate nAChRs in X. laevis oocytes.
Fig. 1. The predicted amino acid sequence and domain structure of X. laevis RIC-3. (a) Alignment of translated sequences for X. laevis, C. kelegans and human RIC-3 showing first and second transmembrane (TM) regions (bold) predicted by TMPred; and the first (bold, highlighted light grey) and second (bold, highlighted dark grey) coiled-coil (CC) domains predicted by COILS. (b) Schematic representation of the domains found in RIC-3 from the three species.
Fig. 2. Concentration-response curves for ACR-16 co-expressed with X. laevis RIC-3. The mean peak current amplitude [normalized to 100 μM acetylcholine (ACh)] was recorded from voltage clamped oocytes 48 h post-injection with 50 ng acr-16/Xenopus ric-3 cRNA at a 1 : 1 ratio (n = 8, n = 4). ACh (closed circles) EC50 = 24 μM (95% CI = 19â30 μM), Hill Slope = 2.177. Nicotine (open circles) EC50 = 22 μM (95% CI = 15â33 μM), Hill Slope = 1.906.
Fig. 3. The effect of varying the amount of X. laevis RIC-3 on the expression of ACR-16 in Xenopus oocytes. (a) The percentage of oocytes expressing detectable currents (> 10nA) in response to 100 μM acetylcholine (ACh) was determined on the experimental weeks in which oocytes from all conditions were screened. The mean percentage expression with the injection of different ratios of acr-16:X. laevis-ric-3 cRNA was as follows; 49 % for 1 : 4, 32 % for 1 : 1, 23 % for 4 : 1 and 7 % for 1 : 0. A One-Way anova with a Tukey's Multiple Comparison Test revealed that the 1 : 4 and 1 : 1 ratio conditions gave significantly more frequent detectable expression than in the 1 : 0 ratio condition (** and * respectively). The 1 : 4 ratio condition was also significantly different to the 4 : 1 condition (*). (b) Mean peak amplitude to 100 μM ACh on ACR-16 expressed in oocytes injected with different ratios of X. laevis ric-3 cRNA to subunit cRNA showing SEM (n = 5, total n for oocytes equal or above 7). Each symbol represents the mean peak amplitude response from a single oocyte on application of 100 μM ACh. The mean results for each condition were as follows; acr-16:X. laevis ric-3 1 : 4 = 662.5 nA, 1 : 1 = 850.7 nA, 1 : 4 = 690 nA, 1 : 0 = 434.4 nA. A one-way anova found that there was no significant difference between the conditions.
Fig. 4. A comparison of the effects of RIC-3 from Xenopus, human and C. elegans on the expression of ACR-16. Example responses to 100 μM acetylcholine (ACh) from oocytes expressing C. elegans ACR-16 (a) in the absence of any RIC-3 and together with RIC-3 from (b) C. elegans, (c) human and (d) X. laevis. (e). The mean peak current amplitudes to 100 μM ACh recorded from voltage clamped oocytes 48 h post-injection with a 1 : 1 ratio of acr-16 cRNA together with ric-3 cRNA from the three species or with none. Each data point represents a single oocyte, and the combined data were obtained from oocytes isolated from at least four different animals. (f) The proportion of the total oocytes tested from which detectable currents (> 10nA) were evoked by the application of 100 μM ACh. ***p = <0.001, **p = <0.01 as compared with the no ric-3 condition.
Fig. 5. A comparison of the effects of RIC-3 from Xenopus, human and C. elegans on the expression of human α7. The maximal current evoked by application of 1 mM ACh was measured in the absence of any exogenous RIC-3 and following co-expression with RIC-3 from C. elegans, human or X. laevis at a 1 : 1 ratio. Each data point represents a single oocyte. No significant differences were detected between the experimental conditions.
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