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.