Visualization of vectorial trafficking of aquaporin‐2 tagged with photoconvertible fluorescence protein in mpkCCD cells (1181.15)

Abstract

AQP2 tagged with green fluorescent protein (GFP‐AQP2) has been used to monitor AQP2 trafficking in kidney cell lines. However, vectorial trafficking of GFP‐AQP2 resembling to that in the principle cells of collecting duct has not been demonstrated in real time in vitro. mEos2 is a photoconvertible fluorescence protein. To visualize AQP2 trafficking, chimeras with mEos2 fused to either the amino‐terminus of AQP2 (Eos‐AQP2) or the carboxyl‐terminus (AQP2‐Eos) via a 17 amino‐acids linker were constructed and expressed in mpkCCD cells on glass‐bottom dishes. Forskolin‐induced apical targeting of AQP2 in real time was monitored using video‐rate confocal microscopy at four dimensions (XYZ‐T). AQP2‐Eos chimeras expressed in mpkCCD cells were localized constitutively to the apical plasma membrane. In contrast, the Eos‐AQP2 chimeras trafficked from intracellular vesicles to the apical membrane in response to forskolin stimulation, indicating similar vectorial trafficking of AQP2 as in collecting duct. Co‐transfection of these two chimeras in 1:1 ratio increased the rate of AQP2 apical accumulation triggered by forskolin (~2‐fold). However, the synergic effect was abolished when the linker was reduced to 8 amino‐acids. These observations suggested that proximity of mEos at the amino‐terminus and the carboxyl‐terminus within the AQP2 tetramer modulates the response to cAMP stimulation.