Aim: 

Aquaporin-4 (AQP4), the major glial water-selective channel in the central nervous system is organized in the plasma membrane in heterotetramers of two different splice variants, called M1 and M23, and forms aggregates called orthogonal arrays of particles (OAPs). When expressed alone, AQP4-M1 does not aggregate in OAPs whereas AQP4-M23 forms large OAPs. We tested the hypothesis that assembly in OAPs increases plasma membrane stability of AQP4.

Methods: 

To study the plasma membrane mobility of AQP4, we used HeLa cells stably transfected with GFP tagged to AQP4-M1 isoform. To induce AQP4 organization in OAPs, these cells were transiently transfected with AQP4-M23 isoform. Lateral mobility was measured by Fluorescence Recovery After Photobleaching (FRAP) technique.

Results: 

FRAP experiments performed on M1 and OAP expressing cells showed that after 6 minutes only in M1 cells there was a fluorescence recovery of the bleached region, which was approximately 90% of the initial fluorescence level. Interestingly, the kinetic of fluorescence recovery was ~ 8 times slower in OAPs expressing cells and the final recovery was approximately only 30% of the initial fluorescence level over 25 min indicating that OAPs are relatively immobile in the plasma membrane.

Conclusion: 

This is the first study showing a biophysical approach to analyze the role of M1 and M23 in the plasma membrane mobility of AQP4 and indicates that AQP4 aggregation in OAPs could be important to increase its level of stability of AQP4 in the plasma membrane.