Gaitán-Peñas H et al. (2018), Expression of LRRC8/VRAC Currents in Xenopus Oo...

XB-ART-54642

Int J Mol Sci 2018 Mar 02;193:. doi: 10.3390/ijms19030719.

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Expression of LRRC8/VRAC Currents in Xenopus Oocytes: Advantages and Caveats.

Gaitán-Peñas H , Pusch M , Estévez R .

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Volume-regulated anion channels (VRACs) play a role in controlling cell volume by opening upon cell swelling. Apart from controlling cell volume, their function is important in many other physiological processes, such as transport of metabolites or drugs, and extracellular signal transduction. VRACs are formed by heteromers of the pannexin homologous protein LRRC8A (also named Swell1) with other LRRC8 members (B, C, D, and E). LRRC8 proteins are difficult to study, since they are expressed in all cells of our body, and the channel stoichiometry can be changed by overexpression, resulting in non-functional heteromers. Two different strategies have been developed to overcome this issue: complementation by transient transfection of LRRC8 genome-edited cell lines, and reconstitution in lipid bilayers. Alternatively, we have used Xenopus oocytes as a simple system to study LRRC8 proteins. Here, we have reviewed all previous experiments that have been performed with VRAC and LRRC8 proteins in Xenopus oocytes. We also discuss future strategies that may be used to perform structure-function analysis of the VRAC in oocytes and other systems, in order to understand its role in controlling multiple physiological functions.

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Species referenced: Xenopus
Genes referenced: lrrc8a

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	Figure 1 Functional expression of LRRC8-mediated VRAC currents in Xenopus oocytes. Currents of single oocytes co-injected with 8A/8E or 8A-VFP/8E-mCherry in response to an iv-pulse protocol in isotonic conditions (Iso) or after a 5 min perfusion with a hypotonic solution (Hypo). On the right, we show the scheme for the injected LRRC8 proteins.
	Figure 2 Biochemical and functional analyses of the currents induced by the expression of 8E-mCherry in Xenopus oocytes. (A) Time course between 2 and 4 days after oocyte injection of the mean values of currents at 60 mV for uninjected oocytes (n ≥ 7), oocytes injected with 8E-mCherry (n ≥ 18), and oocytes injected with 8A-VFP/8E-mCherry (n ≥ 7). Data are from at least two different injections and indicate the mean ± SEM; (B) Western blots against some LRRC8 proteins, using β-tubulin as a loading control. We used protein extracts of (from left to right): collagenase-treated uninjected oocytes “C Un-inj” and oocytes injected with 8A or 8D “hum 8A/8D inj”. Three independent Western-blot experiments to detect Xenopus LRRC8A and two WB experiments to detect Xenopus LRRC8D gave similar results; (C) Comparison of the mean values of current at 60 mV between uninjected oocytes (n = 4) and oocytes co-expressing 8E-mCherry (20 ng of complimentary ribonucleic acid (cRNA)) plus 10 ng of an oligonucleotide sense “x8A sense” (n = 5 at day 3, n = 5 at day 4) or plus 10 ng of an oligo antisense “x8A Asense” (n = 4 at day 3, n = 5 at day 4). Data indicate the mean ± SEM * p < 0.05, p < 0.01, * p < 0.001. Another injection gave similar results.
	Figure 3 Surface analysis of LRRC8 proteins expressed in Xenopus oocytes. Surface expression was examined using a chemiluminescence technique detecting the HA epitope inserted into the first extracellular loop (HAloop) already described [51]. The sequence of LRRC8A with the HA tag was CLPCKWYPYDVPDYAVTKDSC and the sequence of LRRC8E with the HA tag was CLPNHEYPYDVPDYALQENLS (HA tag in italics). The HA tag did not interfere with the proper function of the proteins. In the panel on the left, we show the membrane levels of 8A-VFP, and in the other panel, the levels of 8E-mCherry. We show here a typical experiment with n = 15, 15, and 10 oocytes for the panel on the left, and n = 8, 15, and 9 for the panel on the right.
	Figure 1. Functional expression of LRRC8-mediated VRAC currents in Xenopus oocytes. Currents of single oocytes co-injected with 8A/8E or 8A-VFP/8E-mCherry in response to an iv-pulse protocol in isotonic conditions (Iso) or after a 5 min perfusion with a hypotonic solution (Hypo). On the right, we show the scheme for the injected LRRC8 proteins.
	Figure 2. Biochemical and functional analyses of the currents induced by the expression of 8E-mCherry in Xenopus oocytes. (A) Time course between 2 and 4 days after oocyte injection of the mean values of currents at 60 mV for uninjected oocytes (n ≥ 7), oocytes injected with 8E-mCherry (n ≥ 18), and oocytes injected with 8A-VFP/8E-mCherry (n ≥ 7). Data are from at least two different injections and indicate the mean ± SEM; (B) Western blots against some LRRC8 proteins, using β-tubulin as a loading control. We used protein extracts of (from left to right): collagenase-treated uninjected oocytes “C Un-inj” and oocytes injected with 8A or 8D “hum 8A/8D inj”. Three independent Western-blot experiments to detect Xenopus LRRC8A and two WB experiments to detect Xenopus LRRC8D gave similar results; (C) Comparison of the mean values of current at 60 mV between uninjected oocytes (n = 4) and oocytes co-expressing 8E-mCherry (20 ng of complimentary ribonucleic acid (cRNA)) plus 10 ng of an oligonucleotide sense “x8A sense” (n = 5 at day 3, n = 5 at day 4) or plus 10 ng of an oligo antisense “x8A Asense” (n = 4 at day 3, n = 5 at day 4). Data indicate the mean ± SEM * p < 0.05, p < 0.01, * p < 0.001. Another injection gave similar results.
	Figure 3. Surface analysis of LRRC8 proteins expressed in Xenopus oocytes. Surface expression was examined using a chemiluminescence technique detecting the HA epitope inserted into the first extracellular loop (HAloop) already described [51]. The sequence of LRRC8A with the HA tag was CLPCKWYPYDVPDYAVTKDSC and the sequence of LRRC8E with the HA tag was CLPNHEYPYDVPDYALQENLS (HA tag in italics). The HA tag did not interfere with the proper function of the proteins. In the panel on the left, we show the membrane levels of 8A-VFP, and in the other panel, the levels of 8E-mCherry. We show here a typical experiment with n = 15, 15, and 10 oocytes for the panel on the left, and n = 8, 15, and 9 for the panel on the right.

References [+] :

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