The majority of pathologic mutations in the RS1 gene linked to XLRS pathogenesis regularly result in a deficiency of the encoded protein retinoschisin regardless of the specific type of mutation . To elucidate the consequences of the loss of retinoschisin on the developing retina and thus the pathogenesis of XLRS, the delineation of the retinoschisin interaction partners at the plasma membrane has become a longtime focus in XLRS research. The intracellular beta2-laminin and the extracellular scaffold protein alphaB-crystallin were initially described as interaction partners of retinoschisin . In addition, phosphatidlyserine containing lipid bilayers  and galactose  were proposed to be associated with retinoschisin. Later, a study by Shi and colleagues identified the L-type voltage-gated calcium channels (LTCC) Cav1.3 and Cav1.4 [29, 30]. Eventually, the retinal Na/K-ATPase was demonstrated to directly interact with retinoschisin by Molday and colleagues . Subsequently, our group showed the specific interaction of retinoschisin with the ATP1B2 subunit, whereas the ATP1A3 subunit was exchangeable . Here, we have now added another piece of the puzzle to the growing protein complex associated with retinoschisin.
By co-immunoprecipitation and immunohistochemistry, we demonstrate a physical interaction of subunit ATP1A3 of the retinal Na/K-ATPase complex with Kv channel subunits Kv2.1 and Kv8.2 at the photoreceptor IS. Retinoschisin-deficiency, known to result in XLRS pathology, causes an increasing mislocalization of the retinal Na/K-ATPase and the Kv channel subunits Kv2.1 and Kv8.2 during postnatal retinal development of the XLRS mouse retina. In addition, it is accompanied by a decrease of Kv2.1 and Kv8.2 protein beyond P14, whereas protein levels of the retinal Na/K-ATPase appear unaffected. Of note, we could not observe an effect of retinoschisin-deficiency on the Kv channel mediated potassium ion currents as analyzed in Y-79 cells. It remains to be shown to what extend the pathological findings of each member of the retinoschisin-retinal Na/K-ATPase-Kv channel complex contributes to initial or advanced disease development.
Consistent with our current findings, Kv2.1 and Kv8.2 were shown earlier to be highly expressed in photoreceptor IS, but absent or poorly expressed in other retinal layers [24, 31]. This agrees with our immunohistochemical localization of retinoschisin and the retinal Na/K-ATPase revealing a strong enrichment at the IS of the photoreceptors . It is known that Kv channels together with the Na/K-ATPase play a major role in generating the outward dark current during phototransduction keeping the photoreceptors depolarized and driving the release of glutamate neurotransmitters [32, 33]. In addition, Kv2.1 has a structural function as it mediates spatial and functional coupling of LTCCs and ryanodine receptors in mammalian neurons . Thus, Kv channels are important factors in regulating the electrophysiological integrity in the healthy retina, a process which is obviously disrupted in XLRS [1, 2]. Finally, Kv2.1 was also reported to modulate intracellular signaling [34, 35], yet another process which has been shown to be impaired in XLRS [15, 21, 36].
Following retinoschisin-deficiency, we noticed altered protein expression levels of Kv2.1 and Kv8.2, but not of the retinal Na/K-ATPase as well as an earlier developmental mislocalization of Kv channels compared to the retinal Na/K-ATPase. Interestingly, RT-PCR analysis revealed no changes in mRNA gene expression as the cause for altered protein levels. This is in line with a previous study by Vijayasarathy and colleagues, who used microarray-based genome-wide expression profiling and observed no mRNA expression differences for Kv2.1 and Kv8.2 as well as for Atp1a3 and Atp1b2 in wildtype and retinoschisin-deficient retinae of P12 and P21 old mice . Accordingly, the effect of retinoschisin-deficiency on Kv2.1 and Kv8.2 subunits should be attributed to a posttranslational process. One possibility may be a structural influence on complex integrity due to the absence of the Na/K-ATPase ligand retinoschisin. The integrity of a macromolecular complex is determined by its composition, i.e. the presence of specific ligands/protein binding partners . The stability of the individual complex constituents is also strongly dependent on protein-protein interactions [38–40]. Retinoschisin-deficiency may disturb the formation of the macromolecular Na/K-ATPase-Kv channel complex leading to the observed distinct effects on distribution and total protein amount of the complex components. This is supported by findings in Atp1b2-deficient mice showing that the distribution of Kv2.1 and Kv8.2 in the murine retina is altered due to the lack of the formation of the retinoschisin-Na/K-ATPase complex . Together, the current observations suggest an impaired maintenance of ion homeostasis or defective regulation of intercellular signaling cascades, ultimately initiating the early steps in XLRS pathogenesis.
Aberrant functionality of Kv channels or the Na/K-ATPase have been implicated in various pathological events before. It was reported that Na/K-ATPase binding to AnkB in cardiomyocytes controls the ion homeostasis via regulating the NCX activity in a local domain. Disruption of this interaction resulted in increased calcium sparks and waves, a possible mechanism for arrhythmogenesis in the AnkB syndrome . Also, pathogenic mutations in KCNB1 encoding the Kv2.1 subunit, have been identified in patients with different neurodevelopmental disorders like epilepsy or autism . Further, Kv2.1 knockout mice manifest neuronal and behavioral hyperexcitability , as well as retinal dysfunction. Fortenbach and colleagues showed that the loss of Kv2.1 causes elevated intracellular Ca2+ levels due to elevated Ca2+ influx through cone cyclic nucleotide-gated (CNG) channels which ultimately causes rod degeneration . In the brain, the defective formation of an integrin/alpha5/Kv2.1 macromolecular complex was connected to epilepsy through mechanisms such as abnormal neuronal development . Finally, mutations in KCNV2, encoding Kv8.2, cause the retinal condition cone dystrophy with supernormal rod response (CDSSR) .
Similar to retinoschisin-deficient mice [36, 46, 47], recent studies of Jiang and colleagues as well as of Inamdar and colleagues showed that Kv8.2 knockout mice reveal a significantly higher apoptotic cell count, a thinner retina, and increased microglia occurrence in the subretinal space [31, 48]. Interestingly, the localization of Kv2.1 was found to be unaffected in Kv8.2 knockout mice . Thus, the observed mislocalization of Kv2.1 in our retinoschisin-deficient mice are likely not a general consequence of retinal degeneration, as retinal degeneration also occurs in Kv8.2 knockout mice. This is supported by the fact that the mislocalization of Kv2.1 in the retinoschisin-deficient mouse was detectable as early as P7, long before photoreceptor degeneration at P14 in this mouse model . Therefore, we speculate that the observed mislocalization of the retinal Na/K-ATPase and the Kv-channels Kv2.1 and Kv8.2 may be best explained by an instability of the Na/K-ATPase-Kv-channel complex related to retinoschisin-deficiency. Interestingly, also in Kv8.2 knockout mice the protein level of the Na/K-ATPase was not found to be altered. Of note, the absence of retinoschisin and with this a pathologic distribution of the complex partners may, at least in part, contribute to XLRS manifestations.
Taken together, our data suggest that retinoschisin may act as a crucial interaction partner of an emerging macromolecular complex at the IS of the mammalian photoreceptors by regulating the distribution and stability of the complex and its individual partners. An alteration in the spatial distribution and, consequently, the function of the complex may contribute to clinical symptoms in XLRS. Accordingly, it may be sensible to explore alternative ways to correct the secondary deficits of retinoschisin-deficiency as potential treatment options for XLRS.