Impact of Hybrid and Complex N-Glycans on Cell Surface Targeting of the Endogenous Chloride Cotransporter Slc12a2

Figure 9

Inhibition of N-glycan processing or complex N-glycosylation does not preclude NKCC1 plasma membrane localization but impairs its function. ((a) and (d)) Shown are representative immunoblots experiments showing total endogenous expression levels of NKCC1a in COS7 cells incubated with (DMSO, control) 5–10 μg/mL of kifunensine (KIF, (a)) or 1-2 μg/mL swainsonine (SWN, (d)) for 16 h. Note that the expected bands of NKCC1a, that is, ~130 kDa and ~170 kDa, are detected in control and treated cells whereas additional ckNKCC1-immunoreactive bands centered at ~150 kDa are observed only in protein extracts from KIF/SWN-treated cells. As loading control, immunoblots were developed using antibodies directed against β-actin. ((b) and (e)) Shown are representative immunoblots demonstrating expression of EndoH-sensitive hybrid-type N-glycosylated NKCC1a in total protein extracts of COS7 cells treated for 16 hs with KIF (10 μg/mL) or SWN (2 μg/mL). Note the absence of immunoreactive bands corresponding to NKCC1a ~170 kDa in cell extracts obtained from KIF/SWN-treated cells. ((c) and (f)) Shown are representative immunoblots demonstrating expression of NKCC1a in biotinylated plasma membrane fractions of COS7 cells grown under control conditions or treated for 16 h with KIF or SWN. Protein expression of the cytosolic GAPDH was used to assess the purity of the plasma membrane fractions. (g) Densitometry scanning representing the total cellular N-glycan types of NKCC1 in COS7 cells control or treated for 16 h with KIF (10 μg/mL) or SWN (2 μg/mL). (h) Representative densitometry scanning of NKCC1 immunoblots of plasma membrane fractions obtained from COS7 cells control or treated for 16 h with KIF (10 μg/mL) or SWN (2 μg/mL). (i) Cl⁻ uptake into COS7 cells depleted of endogenous Cl⁻ as a function of time (0–60 min). Uptake is represented as nmol/μg protein [mean ± SEM ()]. Inset: mean Cl⁻ uptake [nmol/μg protein/5 min ± SEM ()] obtained under isotonic conditions (ISO, black bar) or in the presence of BTD 10 μM (ISO + BTD, white bar). (j) Impact of TUN (2 μg/mL), KIF (10 μg/mL), or SWN (1 μg/mL) treatment (16 hs) on the BTD-sensitive component of Cl⁻ uptake into COS7 cells. Results are expressed as nmol/μg protein/5 min ± SEM ().

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