Cristofori-Armstrong B et al. (2015), Xenopus borealis as an alternative source of oo...

XB-ART-51361

Sci Rep 2015 Jan 12;5:14763. doi: 10.1038/srep14763.

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Xenopus borealis as an alternative source of oocytes for biophysical and pharmacological studies of neuronal ion channels.

Cristofori-Armstrong B , Soh MS , Talwar S , Brown DL , Griffin JD , Dekan Z , Stow JL , King GF , Lynch JW , Rash LD .

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For the past 30 years, oocytes from Xenopus laevis have been extensively used to express and characterise ion channels in an easily controlled environment. Here we report the first use of oocytes from the closely related species Xenopus borealis as an alternative expression system for neuronal ion channels. Using the two-electrode voltage-clamp technique, we show that a wide variety of voltage- and ligand-gated ion channels have the same channel properties and pharmacological profiles when expressed in either X. laevis or X. borealis oocytes. Potential advantages of the X. borealis oocytes include a smaller endogenous chloride current and the ability to produce more intense fluorescence signals when studied with voltage-clamp fluorometry. Scanning electron microscopy revealed a difference in vitelline membrane structure between the two species, which may be related to the discrepancy in fluorescence signals observed. We demonstrate that X. borealis oocytes are a viable heterologous system for expression of neuronal ion channels with some potential advantages over X. laevis oocytes for certain applications.

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Species referenced: Xenopus laevis
Genes referenced: asic1 asic2 asic3 kcnh1 kcnh2 nav1 scn2a scn5a

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	Figure 1: Comparison of defolliculated stage V–VI oocytes from X. laevis (left) and X. borealis (right). Oocytes were imaged on an Olympus SZX12 stereomicroscope in ND96 solution.
	Figure 2: Endogenous Ca2+ -activated chloride currents in naïve X. laevis (black) and X. borealis (red) oocytes. Representative families of whole-cell currents elicited by steps from −120 mV to +100 mV in 20-mV increments in oocytes from (a) X. laevis and (b) X. borealis (holding potential of −100 mV). (c) Peak amplitude as measured at the end of a 2 s pulse to +100 mV. A significant difference was observed between the two species (unpaired t-test, P = 8.3 × 10−5). Each data point indicates recording from a single oocyte (n = 22). Several oocytes were used from each of five individual frogs of each species. Data are presented as mean (dashed line) and 95% confidence intervals.
	Figure 3: Voltage-dependent properties of KV and NaV channels expressed in X. laevis (black) and X. borealis (red) oocytes. (a) Current-voltage relationship showing activation of KV10.1 channels (n = 12). (b) KV11.1 tail current plotted as a function of voltage to show activation properties (n = 9–12). The normalised deduced conductance (G)-voltage relationships for (c) NaV1.2, (d) NaV1.5, and (e) NaV1.7 (n = 12–17). (f) The effect of 300 nM ProTx-I on current evoked by a depolarisation to –15 mV from a holding potential of −80 mV (n = 8–9). There is no evidence of a difference between the two species (unpaired t-test, P > 0.05). Error bars indicate 95% confidence intervals.
	Figure 4: The effect of activating and antagonist ligands on different ASIC and GABAAR subtypes expressed in X. laevis (black) and X. borealis (red) oocytes. pH-dependence of steady-state desensitisation (open symbols, dashed lines) and activation (closed symbols, solid lines) of (a) ASIC1a, (b) ASIC1b, (c) ASIC2a, and (d) ASIC3 (n = 9–12). (e) Concentration-effect curve for inhibition of rat ASIC1a by Pc1a (n = 9–12). (f) Concentration-effect curve for inhibition of rat ASIC3 by APETx2 (n = 9). Concentration-effect curves for GABA activation of the (g) α1β2γ2L, (h) α5β2γ2L, and (i) α5β3γ2L subtypes of GABAARs (n = 9–10). Error bars indicate 95% confidence intervals.
	Figure 5: Effect of protease treatment on fluorescence signals (ΔF) from X. laevis and X. borealis oocytes expressing the α1N203C GlyR with current induced by 10 μM glycine. (a) Representative current and fluorescence traces show a significantly greater ΔF in untreated X. laevis than X. borealis oocytes. (b) A significant increase in ΔF was obtained after 1 min treatment with protease for X. borealis, but not X. laevis, oocytes. Traces from A and B represent separate oocytes. (c) ΔF of X. laevis and X. borealis oocytes in all conditions tested (n = 7). X. laevis oocytes were not amenable to protease treatment for >1 min as this damaged membrane integrity. P values were calculated in comparison to untreated oocytes from each species using an unpaired t-test with Welch’s correction (P < 0.01, *P < 0.001). Error bars indicate 95% confidence intervals.
	Figure 6: Expression of GlyRs in X. laevis (black) and X. borealis (red) oocytes as determined by voltage-clamp fluorometry. (a) Example ΔF traces from oocytes expressing labelled α1N203C GlyR. (b) and (c) Normalised glycine concentration-effect curves for both current (closed symbols, solid lines) and fluorescence (open symbols, dashed lines) of MTS-TAMRA labelled α1N203C GlyR and MTSR labelled α1R271C GlyR using voltage-clamp fluorometry (n = 4–6). Error bars indicate 95% confidence intervals.
	Figure 7: Protease treatment affects dye accessibility to channels in the plasma membrane. Effect of MTS-TAMARA labelling on the background fluorescence of X. laevis (black) and X. borealis (red) oocytes expressing α1N203C GlyR prior to (untreated, circles) and following protease exposure (1 min treated, squares) (n = 7). Fluorescence readings were normalised to oocytes that had been protease treated then labelled. Error bars indicate 95% confidence intervals.
	Figure 8: SEM images of defolliculated X. laevis and X. borealis stage V–VI oocytes prior to and following protease treatment. Oocytes from X. laevis (a–f) and X. borealis (g–l) were viewed by SEM following treatment with protease for 1 min (b,e,h,k) and 1.5 mins (c,f,i,l). At higher magnifications the typically contoured surface and pores of the vitelline membrane of untreated X. laevis (d) and X. borealis (j) become apparent, scale bar = 10 μm. One minute protease treatment of oocytes affects the vitelline membrane of X. laevis (e) and X. borealis (k) differently, scale bar = 10 μm. Microvilli projections on the plasma membranes of X. laevis (f) and X. borealis (l) oocytes show different morphology and density, scale bar = 2 μm. Each panel represents a separate oocyte.
	Figure 1. Comparison of defolliculated stage V–VI oocytes from X. laevis (left) and X. borealis (right).Oocytes were imaged on an Olympus SZX12 stereomicroscope in ND96 solution.
	Figure 2. Endogenous Ca2+ -activated chloride currents in naïve X. laevis (•) and X. borealis () oocytes.Representative families of whole-cell currents elicited by steps from −120 mV to +100 mV in 20-mV increments in oocytes from (a) X. laevis and (b) X. borealis (holding potential of −100 mV). (c) Peak amplitude as measured at the end of a 2 s pulse to +100 mV. A significant difference was observed between the two species (unpaired t-test, P = 8.3 × 10−5). Each data point indicates recording from a single oocyte (n = 22). Several oocytes were used from each of five individual frogs of each species. Data are presented as mean (dashed line) and 95% confidence intervals.
	Figure 3. Voltage-dependent properties of KV and NaV channels expressed in X. laevis (•) and X. borealis () oocytes.(a) Current-voltage relationship showing activation of KV10.1 channels (n = 12). (b) KV11.1 tail current plotted as a function of voltage to show activation properties (n = 9–12). The normalised deduced conductance (G)-voltage relationships for (c) NaV1.2, (d) NaV1.5, and (e) NaV1.7 (n = 12–17). (f) The effect of 300 nM ProTx-I on current evoked by a depolarisation to –15 mV from a holding potential of −80 mV (n = 8–9). There is no evidence of a difference between the two species (unpaired t-test, P > 0.05). Error bars indicate 95% confidence intervals.
	Figure 4. The effect of activating and antagonist ligands on different ASIC and GABAAR subtypes expressed in X. laevis (•) and X. borealis () oocytes.pH-dependence of steady-state desensitisation (open symbols, dashed lines) and activation (closed symbols, solid lines) of (a) ASIC1a, (b) ASIC1b, (c) ASIC2a, and (d) ASIC3 (n = 9–12). (e) Concentration-effect curve for inhibition of rat ASIC1a by Pc1a (n = 9–12). (f) Concentration-effect curve for inhibition of rat ASIC3 by APETx2 (n = 9). Concentration-effect curves for GABA activation of the (g) α1β2γ2L, (h) α5β2γ2L, and (i) α5β3γ2L subtypes of GABAARs (n = 9–10). Error bars indicate 95% confidence intervals.
	Figure 5. Effect of protease treatment on fluorescence signals (ΔF) from X. laevis and X. borealis oocytes expressing the α1N203C GlyR with current induced by 10 μM glycine.(a) Representative current and fluorescence traces show a significantly greater ΔF in untreated X. laevis than X. borealis oocytes. (b) A significant increase in ΔF was obtained after 1 min treatment with protease for X. borealis, but not X. laevis, oocytes. Traces from A and B represent separate oocytes. (c) ΔF of X. laevis and X. borealis oocytes in all conditions tested (n = 7). X. laevis oocytes were not amenable to protease treatment for >1 min as this damaged membrane integrity. P values were calculated in comparison to untreated oocytes from each species using an unpaired t-test with Welch’s correction (P < 0.01, *P < 0.001). Error bars indicate 95% confidence intervals.
	Figure 6. Expression of GlyRs in X. laevis (•) and X. borealis () oocytes as determined by voltage-clamp fluorometry.(a) Example ΔF traces from oocytes expressing labelled α1N203C GlyR. (b) and (c) Normalised glycine concentration-effect curves for both current (closed symbols, solid lines) and fluorescence (open symbols, dashed lines) of MTS-TAMRA labelled α1N203C GlyR and MTSR labelled α1R271C GlyR using voltage-clamp fluorometry (n = 4–6). Error bars indicate 95% confidence intervals.
	Figure 7. Protease treatment affects dye accessibility to channels in the plasma membrane.Effect of MTS-TAMARA labelling on the background fluorescence of X. laevis (black) and X. borealis (red) oocytes expressing α1N203C GlyR prior to (untreated, circles) and following protease exposure (1 min treated, squares) (n = 7). Fluorescence readings were normalised to oocytes that had been protease treated then labelled. Error bars indicate 95% confidence intervals.
	Figure 8. SEM images of defolliculated X. laevis and X. borealis stage V–VI oocytes prior to and following protease treatment.Oocytes from X. laevis (a–f) and X. borealis (g–l) were viewed by SEM following treatment with protease for 1 min (b,e,h,k) and 1.5 mins (c,f,i,l). At higher magnifications the typically contoured surface and pores of the vitelline membrane of untreated X. laevis (d) and X. borealis (j) become apparent, scale bar = 10 μm. One minute protease treatment of oocytes affects the vitelline membrane of X. laevis (e) and X. borealis (k) differently, scale bar = 10 μm. Microvilli projections on the plasma membranes of X. laevis (f) and X. borealis (l) oocytes show different morphology and density, scale bar = 2 μm. Each panel represents a separate oocyte.

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