Electrical Communication and its Physiological Relevance in Retinal Pigment Epithelium

Retinal pigment epithelium (RPE) is a tissue that preserves the health and functionality of its closely associated neural tissue, the retina. Many of the essential functions of RPE, including the renewal of light-sensing retinal photoreceptors, are regulated by ion channels. Yet, the involved ionic mechanisms, the extent of membrane potential dynamics, and the intercellular communication are not entirely understood. In this thesis, I studied the voltage-gated ion channels and electrical coupling of RPE in both human embryonic stem cell-derived and mouse RPE.

Voltage-gated sodium channels (NaV), while best known for their role in action potential generation, are expressed in several non-excitable cell types such as macrophages and astrocytes. Yet, these channels had not been considered to exist in native RPE, although they had occasionally been detected in cell culture. This thesis demonstrates that stem cell-derived and mouse RPE exhibit several subtypes of NaV channels and that their earlier dismissal was due to cell isolation procedures. Our electrophysiological recordings showed that these identified NaV channels are functional. The main channel subtypes NaV1.4–NaV1.6 and NaV1.8 were found to localize in the cell-cell junctions or apical membrane in RPE.

As the conventional method to carry out electrophysiological recordings in RPE is to use single cells, the electrical connectivity had not been characterized in mammalian RPE, despite the importance of gap junctions in ocular development. In this thesis, we showed that the major connexin (Cx) isoform was Cx43 which was found to form both gap junctions and apical hemichannels. The electrophysiological recordings demonstrated that the electrical connectivity was relatively low despite the extensive network of gap junctions in RPE. Yet, it was modifiable by gap junction blockers or by inhibiting a specific kinase known as cyclin-dependent kinase 5 (Cdk5).

The significance of NaV channels and gap junctions to RPE physiology was investigated by focusing on the renewal of photoreceptor outer segments, where phagocytosis by RPE plays a key role. The results demonstrated that NaV subtypes NaV1.4 and NaV1.8 localize with the outer segments during phagocytosis. Moreover, inhibiting the activity of NaV channels with pharmacological modulators or short hairpin RNA (shRNA) significantly impaired phagocytosis efficiency.

Furthermore, Nav channels were found to localize to the forming phagocytic cups in the apical membrane and the ingested phagosomes together with an endosomal marker Rab7. The results obtained in this thesis imply that NaV channels have versatile roles in phagocytosis.

In addition to NaV channels, Cx43 localized adjacent to outer segments during phagocytosis, and the results indicate that gap junctions are internalized during the process. This translocation of gap junctions was shown to be regulated by phosphorylation, particularly by kinases such as Cdk5 and protein kinase C. The results obtained in this thesis imply that Cx43 is involved in the formation of phagocytic cups. As phagocytosis is known to be under circadian control, and Cdk5 has previously been shown to regulate this cycle, it is plausible that Cdk5 helps to control the rhythm of photoreceptor renewal.

Our results highlight the complexity of RPE physiology and its ion channel machinery. The findings add to the growing body of evidence demonstrating that NaV channels' role is much more diverse than action potential generation. The results show that RPE can generate fast changes in voltage and rapidly modify its cell-cell connectivity across the epithelium. Gaining a deeper understanding of the involvement of ionic mechanisms in phagocytosis could help us to understand the phagocytosis pathway both in the healthy and diseased eye. Ultimately, this work highlights that RPE's role in its interaction with the neural retina is far more active than was previously thought.