Abstract
Purpose :
Retinal pigment epithelium (RPE) maintains the functionality of the light sensing photoreceptors by facilitating the daily renewal of their outer segments through phagocytosis. The phagocytosis process is complex and under precise regulation. Emerging experimental data show the involvement of voltage-gated ion channels in the early phases of phagocytosis suggesting changes in membrane potential during the process. However, to date, the role of these channels in phagocytosis has been studied primarily by cell and molecular biology methodology resulting in an incomplete understanding of the concurrent electrical signals. The aim of this study was to measure changes in the electrical properties of RPE cells before and after activation of phagocytosis.
Methods :
We used human induced pluripotent stem cell derived RPE lines expressing endogenously GFP-tagged β-actin. Changes in membrane potential were measured using whole cell patch clamp technique in perforated patch configuration. Phagocytosis was initiated in a controlled way by using glass micropipette coated with fluorescently labelled photoreceptor outer segment (POS) particles. The pipette was brought in contact with the recorded cell and membrane potential was continuously monitored during particle binding and engulfment. After the measurements, both glass pipettes were moved away, and fluorescence live-cell imaging was used to track the ongoing phagocytosis process by following changes in GFP-tagged β-actin and pre-labelled POS particles.
Results :
Utilizing conventional patch clamp technique, we created an approach to monitor changes in cellular electrical properties during phagocytosis. With the method, binding and uptake of POS particles were possible to perform in a controlled manner in the recorded cell. The recordings did not disturb the cell’s normal physiology and phagocytosis continued while monitoring the cell’s electrical properties with patch clamp technique. Excitingly, our results reveal changes in membrane potential during phagocytosis.
Conclusions :
Our method enabled the measurements of cellular electrical properties in RPE during POS phagocytosis. Our data suggest that particle binding and uptake involve changes in RPE membrane potential. The method therefore provide means to reach a mechanistic understanding on the role of voltage-gated ion channels in RPE to the phagocytosis of photoreceptor outer segments.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.