Abstract
Purpose :
Maintenance of Na+ and K+ gradients by the Na,K-ATPase is crucial for proper cellular function and survival. Na,K-ATPase is a heterodimer, made up of a catalytic a and b subunit, integrated into the plasma membrane. In photoreceptors, the Na,K-ATPase maintains the photocurrent required for transduction of visual signals to downstream neurons. Localized to the inner segment, photoreceptor Na,K-ATPase is comprised predominantly of ATP1β2 and ATP1α3. Genetic ablation of Atp1b2 results in rapid degeneration of photoreceptors, and loss of retinal ATP1α3 expression. This phenotype is not rescued by substitution of ATP1β2 with ATP1β1, highlighting a unique requirement for ATP1β2 in photoreceptor function and survival. The specific role of ATP1β2 in photoreceptor neurons remains unclear and is the focus of this study. Our lab has identified that ATP1β2 undergoes palmitoylation, a reversible, post-translational lipid modification.
Methods :
Palmitoylation of ATP1β2 was assayed by acyl resin-assisted capture (acyl-RAC) in wild-type murine retina. Palmitoylation was further confirmed in mammalian cell culture by metabolic labeling with 17-ODYA, a palmitoyl chemical analog. Palmitoylation prediction software CSS-Palm was used to design the mutant ATP1β2-C10S constructs. HEK293 cells were transiently transfected with wild-type or mutant mouse ATP1β2 constructs. Expression and stability of ATP1β2 and ATP1α3 was monitored using immunoblot. Association between wild-type or mutant ATP1β2 and ATP1α3 was analyzed using co-immunoprecipitation, while trafficking and membrane association was assessed using membrane fractionation and immunocytochemistry.
Results :
Using acyl-RAC, we have determined that retinal ATP1β2 is palmitoylated. CSS-Palm predicts the 10th amino acid (Cys-10) in ATP1β2 to be palmitoylated, which is unique to the β2-subunit of the Na,K-ATPase. 17-ODYA labeling in HEK293 cells further confirms this cysteine residue to be palmitoylated, as we observe that a cysteine to serine (C10S) mutation results in loss of palmitoylation of ATP1β2. Additionally, we see that wild-type ATP1β2 and ATP1α3 are enriched at the plasma membrane, while mutant ATP1β2 and ATP1α3 mislocalize to the cytosol in cell culture.
Conclusions :
Our findings suggest that palmitoylation of ATP1β2 plays a major role in its association and ATP1α3, as well as proper trafficking of this heterodimeric enzyme to the plasma membrane.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.