Identification of a <scp>VxP</scp> Targeting Signal in the Flagellar Na<sup>+</sup>/K<sup>+</sup>‐<scp>ATPase</scp>

Laird, Joseph G.; Pan, Yuan; Modestou, Modestos; Yamaguchi, David M.; Song, Hailong; Sokolov, Maxim; Baker, Sheila A.

doi:10.1111/tra.12332

Cited by 6 publications

(5 citation statements)

References 76 publications

(159 reference statements)

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“…Next, we investigated (co-)localization of the retinal Na/K-ATPase and Kv channel subunits Kv2.1 and Kv8.2 at the photoreceptor IS via immunohistochemistry of murine retinal cryosections. Consistent with the current literature [ 26 – 29 , 38 ], the retinal Na/K-ATPase, retinoschisin and Kv2.1 were localized to the plasma membrane of the IS. Kv8.2 signals had a punctuate appearance, and, while labelling of the IS still is obvious, membrane localization could not be confirmed in this experiment.…”

Section: Resultssupporting

confidence: 91%

Retinoschisin and novel Na/K-ATPase interaction partners Kv2.1 and Kv8.2 define a growing protein complex at the inner segments of mammalian photoreceptors

Schmid

Wurzel

Wetzel

et al. 2022

Cell. Mol. Life Sci.

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The RS1 gene on Xp 22.13 encodes retinoschisin which is known to directly interact with the retinal Na/K-ATPase at the photoreceptor inner segments. Pathologic mutations in RS1 cause X-linked juvenile retinoschisis (XLRS), a hereditary retinal dystrophy in young males. To further delineate the retinoschisin-Na/K-ATPase complex, co-immunoprecipitation was performed with porcine and murine retinal lysates targeting the ATP1A3 subunit. This identified the voltage-gated potassium (Kv) channel subunits Kv2.1 and Kv8.2 as direct interaction partners of the retinal Na/K-ATPase. Colocalization of the individual components of the complex was demonstrated at the membrane of photoreceptor inner segments. We further show that retinoschisin-deficiency, a frequent consequence of molecular pathology in XLRS, causes mislocalization of the macromolecular complex during postnatal retinal development with a simultaneous reduction of Kv2.1 and Kv8.2 protein expression, while the level of retinal Na/K-ATPase expression remains unaffected. Patch-clamp analysis revealed no effect of retinoschisin-deficiency on Kv channel mediated potassium ion currents in vitro. Together, our data suggest that Kv2.1 and Kv8.2 together with retinoschisin and the retinal Na/K-ATPase are integral parts of a macromolecular complex at the photoreceptor inner segments. Defective compartmentalization of this complex due to retinoschisin-deficiency may be a crucial step in initial XLRS pathogenesis.

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Section: Resultssupporting

confidence: 91%

Retinoschisin and novel Na/K-ATPase interaction partners Kv2.1 and Kv8.2 define a growing protein complex at the inner segments of mammalian photoreceptors

Schmid

Wurzel

Wetzel

et al. 2022

Cell. Mol. Life Sci.

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“…DNA expression vectors were generated by standard methods combining PCR, DNA recombination, and site-directed mutagenesis. The expression vector containing the Xenopus opsin promotor (XOP) followed by GFP-NKAa3 (Laird et al, 2015) was modified. The vector contained an AgeI site upstream of GFP-NKAa.…”

Section: Methodsmentioning

confidence: 99%

RPE Cells Engulf Microvesicles Secreted by Degenerating Rod Photoreceptors

Ropelewski

Imanishi

2020

eNeuro

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Rhodopsin is mislocalized to the inner segment plasma membrane (IS PM) in various blinding disorders including autosomal-dominant retinitis pigmentosa caused by class I rhodopsin mutations. In these disorders, rhodopsin-laden microvesicles are secreted into the extracellular milieu by afflicted photoreceptor cells. Using a Xenopus laevis model expressing class I mutant rhodopsin or Na 1 /K 1-ATPase (NKA) fused to Dendra2, we fluorescently labeled the microvesicles and found retinal pigment epithelial (RPE) cells are capable of engulfing microvesicles containing rhodopsin. A unique sorting mechanism allows class I mutant rhodopsin, but not NKA, to be packaged into the microvesicles. Under normal physiological conditions, NKA is not shed as Significance Statement Rhodopsin mislocalization is a common cause of photoreceptor degeneration in inherited blinding disorders. In this study, we describe a novel mechanism of removing rhodopsin molecules from inner segment plasma membrane (IS PM). Mislocalized rhodopsin is packaged into microvesicles, which are secreted into the interphotoreceptor space and cleared via engulfment by retinal pigment epithelial (RPE) cells. While IS PM-mislocalized rhodopsin is specifically packaged into microvesicles, Na 1 /K 1-ATPase a-subunit, an IS PM resident protein, was not sorted into vesicles under either pathologic or normal physiological conditions. Interaction between photoreceptor and RPE cells is critical for maintaining visual function, and its alteration can lead to compromised vision. This study provides novel insights into photoreceptor-RPE cell interaction in inherited blinding disorders.

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“…DNA expression vectors were generated by standard methods combining PCR, DNA recombination, and site-directed mutagenesis. The expression vector containing the Xenopus opsin promotor (XOP) followed by GFP-sodium-potassium ATPase ␣-subunit (NKA␣; Laird et al, 2015) was a kind gift from Dr. Sheila Baker (University of Iowa). The vector contained an AgeI site upstream of GFP-NKA␣.…”

Section: Methodsmentioning

confidence: 99%

Disrupted Plasma Membrane Protein Homeostasis in a Xenopus Laevis Model of Retinitis Pigmentosa

Ropelewski

Imanishi

2019

J. Neurosci.

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Rhodopsin mislocalization is frequently observed in retinitis pigmentosa (RP) patients. For example, class I mutant rhodopsin is deficient in the VxPx trafficking signal, mislocalizes to the plasma membrane (PM) of rod photoreceptor inner segments (ISs), and causes autosomal dominant RP. Mislocalized rhodopsin causes photoreceptor degeneration in a manner independent of light-activation. In this manuscript, we took advantage of Xenopus laevis models of both sexes expressing wild-type human rhodopsin or its class I Q344ter mutant fused to Dendra2 fluorescent protein to characterize a novel light-independent mechanism of photoreceptor degeneration caused by mislocalized rhodopsin. We found that rhodopsin mislocalized to the PM is actively internalized and transported to lysosomes where it is degraded. This degradation process results in the downregulation of a crucial component of the photoreceptor IS PM: the sodiumpotassium ATPase ␣-subunit (NKA␣). The downregulation of NKA␣ is not because of decreased NKA␣ mRNA, but due to cotransport of mislocalized rhodopsin and NKA␣ to lysosomes or autophagolysosomes. In a separate set of experiments, we found that class I mutant rhodopsin, which causes NKA␣ downregulation, also causes shortening and loss of rod outer segments (OSs); the symptoms frequently observed in the early stages of human RP. Likewise, pharmacological inhibition of NKA␣ led to shortening and loss of rod OSs. These combined studies suggest that mislocalized rhodopsin leads to photoreceptor dysfunction through disruption of the PM protein homeostasis and compromised NKA␣ function. This study unveiled a novel role of lysosome-mediated degradation in causing inherited disorders manifested by mislocalization of ciliary receptors. Significance StatementRetinal ciliopathy is the most common form of inherited blinding disorder frequently manifesting rhodopsin mislocalization. Our understanding of the relationships between rhodopsin mislocalization and photoreceptor dysfunction/degeneration has been far from complete. This study uncovers a hitherto uncharacterized consequence of rhodopsin mislocalization: the activation of the lysosomal pathway, which negatively regulates the amount of the sodium-potassium ATPase (NKA␣) on the inner segment plasma membrane. On the plasma membrane, mislocalized rhodopsin extracts NKA␣ and sends it to lysosomes where they are co-degraded. Compromised NKA␣ function leads to shortening and loss of the photoreceptor outer segments as observed for various inherited blinding disorders. In summary, this study revealed a novel pathogenic mechanism applicable to various forms of blinding disorders caused by rhodopsin mislocalization.

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Identification of a VxP Targeting Signal in the Flagellar Na⁺/K⁺‐ATPase

Cited by 6 publications

References 76 publications

Retinoschisin and novel Na/K-ATPase interaction partners Kv2.1 and Kv8.2 define a growing protein complex at the inner segments of mammalian photoreceptors

Retinoschisin and novel Na/K-ATPase interaction partners Kv2.1 and Kv8.2 define a growing protein complex at the inner segments of mammalian photoreceptors

RPE Cells Engulf Microvesicles Secreted by Degenerating Rod Photoreceptors

Disrupted Plasma Membrane Protein Homeostasis in a Xenopus Laevis Model of Retinitis Pigmentosa

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Identification of a VxP Targeting Signal in the Flagellar Na+/K+‐ATPase

Cited by 6 publications

References 76 publications

Retinoschisin and novel Na/K-ATPase interaction partners Kv2.1 and Kv8.2 define a growing protein complex at the inner segments of mammalian photoreceptors

Retinoschisin and novel Na/K-ATPase interaction partners Kv2.1 and Kv8.2 define a growing protein complex at the inner segments of mammalian photoreceptors

RPE Cells Engulf Microvesicles Secreted by Degenerating Rod Photoreceptors

Disrupted Plasma Membrane Protein Homeostasis in a Xenopus Laevis Model of Retinitis Pigmentosa

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Identification of a VxP Targeting Signal in the Flagellar Na⁺/K⁺‐ATPase