Lopez-Rodriguez A and Holmgren M (2012), Restoration of proper trafficking to the cell s...

XB-ART-46907

PLoS One 2012 Jan 01;710:e47693. doi: 10.1371/journal.pone.0047693.

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Restoration of proper trafficking to the cell surface for membrane proteins harboring cysteine mutations.

Lopez-Rodriguez A, Holmgren M.

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A common phenotype for many genetic diseases is that the cell is unable to deliver full-length membrane proteins to the cell surface. For some forms of autism, hereditary spherocytosis and color blindness, the culprits are single point mutations to cysteine. We have studied two inheritable cysteine mutants of cyclic nucleotide-gated channels that produce achromatopsia, a common form of severe color blindness. By taking advantage of the reactivity of cysteine's sulfhydryl group, we modified these mutants with chemical reagents that attach moieties with similar chemistries to the wild-type amino acids' side chains. We show that these modifications restored proper delivery to the cell membrane. Once there, the channels exhibited normal functional properties. This strategy might provide a unique opportunity to assess the chemical nature of membrane protein traffic problems.

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Species referenced: Xenopus
Genes referenced: camp cnga1

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	Figure 2. Time course of cell surface expression for R272C CNGA1-GFP channels exposed to MTSEA.A. Representative confocal images of one oocyte before (T0) and after 2 mM MTSEA exposure. Media with fresh MTSEA was exchanged every 30 min. After ∼6 hrs of MTSEA treatment, GFP fluorescence was detectable at the oocyte’s surface. B. Plot shows the time course of cell surface fluorescence detection in six different oocytes.
	Figure 3. Functional characterization of CNGA1-GFP channels at the cell surface.A, B & C. Cysteine-less CNGA1-GFP channels. A, Ionic currents in the presence of saturating [cGMP] (2 mM). cGMP-activated currents shown were acquired in response to 100 ms voltage steps from −80 to +80 mV (every 20 mV) from a holding potential of 0 mV. B. Dose response for cGMP at +60 mV. Solid line represents a normalized Hill equation fit to the data. The best fit parameter values for K½ was 19±1 µM, and for the hill coefficient (n) was 2.1±0.2. Average values for K½ and n were 24±3 µM and 2.16±0.46, respectively (n = 4 oocytes). C. Efficacies for different agonists. Wild type CNGA1 channels displayed larger than 95% maximal probability of opening with saturated concentrations of cGMP, less with cIMP, and much less with cAMP. Nucleotide-activated current records shown were obtained from the same excised inside out patch with saturating concentrations of cGMP (2 mM; black), cIMP (16 mM; red) and cAMP (16 mM; blue). D, E & F. Rescued R272C CNGA1-GFP channels. D. Ionic current carried by MTSEA-modified R272C channels. In response to a comparable experimental protocol as in A, rescued channels were able to conduct ionic current with similar properties as wild type channels. E. Dose response of modified R272C channels at the cell surface for cGMP. Solid line corresponds to a normalized Hill equation fit to the data. The best fit parameter values for K½ and n were 43.4±2.5 µM and 1.9±0.3, respectively. Average values for K½ and n were 59±10 µM and 1.48±0.22, respectively (n = 14 oocytes). F. Agonist efficacies for rescued R272C mutant channels. Nucleotide-activated current records shown were acquired from the same patch, using the equivalent agonist concentration as in C. Similar observations were made in 3 different patches.
	Figure 4. Cell surface expression of CNGA1-GFP channels.A. Representative Western blot of total CNGA1-GFP protein (TP) and biotinylated CNGA1-GFP cell surface protein (SP). WT, R272C, R272C+MTSEA denote cysteine-less CNGA1-GFP, mutant channels that were not treated with MTSEA and mutant channels that were modified by MTSEA, respectively. An expected band of ∼106 kDa for the deglycosylated wild-type GFP tagged channel was detected by chemiluminiscence using a GFP antibody. No signal was detected in non injected oocytes. B. Densitometry analysis of the bands normalized to TP (n = 3 different oocytes batches).
	Figure 5. Y176C CNGA1-GFP mutant channels.A. Confocal image of an oocyte injected with cRNA encoding for Y176C CNGA1-GFP channels. We never detected any signs of proper trafficking in >100 oocytes by standard fluorescence microscopy, suggesting that these channels do not reach the cell surface. B. MTSHB treatment recovers proper trafficking of Y176C mutant channels to the cell surface. Confocal image shows GFP fluorescent signal at the cell surface of an oocyte expressing Y176C mutant channels. MTSHB was injected ∼ 12 h before acquiring the image (n >25 oocytes). C. Ionic currents in the presence of a saturating cGMP concentration (2 mM), in response to the same voltage protocol as described in Fig. 3A. D. Dose response for cGMP at +60 mV. Solid line represents a normalized Hill equation fit to the data. The best fit parameter values for K½ and n were 16±1 µM and 1.30±0.05, respectively. From a total of 8 oocytes, the average values for K½ and n were 16±3 µM and 1.6±0.1, respectively. E. Agonist efficacies of rescued Y176C mutant channels. Nucleotide-activated current records shown in the presence of saturating concentrations of each agonist (same as Fig. 3C) were acquired from the same excised patch. Rescued Y176C channels showed a similar efficacy pattern as cysteine-less CNGA1-GFP channels. Similar observations were observed in 4 patches.

References [+] :

Aggarwal, Contribution of the S4 segment to gating charge in the Shaker K+ channel. 1996, Pubmed, Xenbase