CHRM1 mutant P380L presents an altered traffic to the plasma membrane
CHRM1 is a protein whose molecular weight as a monomer is 52 kDa, but the mature protein increases its molecular weight due to glycosylation. To study the effect of the mutation p.Pro380Leu, wild-type CHRM1 or mutant were expressed into HEK293T cells and protein expression levels were analysed (Figure 2A ). The mutant showed a different protein pattern band compared to wild-type protein, with a reduction of protein band 1 (wild-type: 71 ± 14 %, P380L: 14 ± 2 % of total protein, n= 5), corresponding to the mature glycosylated form, and an increase of bands 2 and 3 (wild-type: 19 ± 10 %, P380L: 64 ± 26% of total protein, n= 5), corresponding to immature forms, as revealed by glycosidase treatment (not shown). Then, we reasoned that mutation P380L impairs the correct maturation of CHRM1 protein.
To verify this biochemical data, we co-transfected HeLa cells with CHRM1 wild-type or mutant plus PH-GFP, a fluorescent probe that labels the plasma membrane (Figure 2B ). Whereas CHRM1 was detected mainly at the plasma membrane, predominantly colocalizing with PH-GFP (yellow staining) (Pearson’s correlation coefficient: Rr = 0.8 ± 0.02, n= 3 / 65 cells), the mutant was almost exclusively detected in intracellular compartments (Figure 2B), showing less degree of colocalization with PH-GFP (Rr = 0.3 ± 0.02, n = 3 / 45 cells). Thus, immunofluorescence studies confirmed that the mutation reduced surface expression of the CHRM1 protein.