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Heteromeric KV2/KV8.2 Channels Mediate Delayed Rectifier Potassium Currents in Primate Photoreceptors

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Heteromeric KV2/KV8.2 Channels Mediate Delayed Rectifier Potassium Currents in Primate Photoreceptors 3414 • The Journal of Neuroscience, April 4, 2018 • 38(14):3414–3427 Systems/Circuits Heteromeric KV2/KV8.2 Channels Mediate Delayed Rectifier Potassium Currents in Primate Photoreceptors Jacqueline Gayet-Primo,1 Daniel B. Yaeger,3 Roupen A. Khanjian,3 and XTeresa Puthussery1,2,3 1School of Optometry, 2Helen Wills Neuroscience Institute, University of California, Berkeley, Berkeley, California 94720, and 3Casey Eye Institute, Oregon Health & Science University, Portland, Oregon 97239 Silent voltage-gated potassium channel subunits (KVS) interact selectively with members of the KV2 channel family to modify their functional properties. The localization and functional roles of these silent subunits remain poorly understood. Mutations in the KVS subunit, KV8.2 (KCNV2), lead to severe visual impairment in humans, but the basis of these deficits remains unclear. Here, we examined the localization, native interactions, and functional properties of KV8.2-containing channels in mouse, macaque, and human photore- ceptors of either sex. In human retina, KV8.2 colocalized with KV2.1 and KV2.2 in cone inner segments and with KV2.1 in rod inner segments. KV2.1 and KV2.2 could be coimmunoprecipitated with KV8.2 in retinal lysates indicating that these subunits likely interact directly. Retinal KV2.1 was less phosphorylated than cortical KV2.1, a difference expected to alter the biophysical properties of these channels. Using voltage-clamp recordings and pharmacology, we provide functional evidence for Kv2-containing channels in primate rods and cones. We propose that the presence of KV8.2, and low levels of KV2.1 phosphorylation shift the activation range of KV2 channels toalignwiththeoperatingrangeofrodandconephotoreceptors.OurdataindicatearoleforKV2/KV8.2channelsinhumanphotoreceptor function and suggest that the visual deficits in patients with KCNV2 mutations arise from inadequate resting activation of KV channels in rod and cone inner segments. Key words: cones; ion channels; potassium channels; retina; rods Significance Statement Mutationsinavoltage-gatedpotassiumchannelsubunit,KV8.2,underlieablindinginheritedphotoreceptordystrophy,indicating an important role for these channels in human vision. Here, we have defined the localization and subunit interactions of KV8.2 channels in primate photoreceptors. We show that the KV8.2 subunit interacts with different Kv2 channels in rods and cones, giving rise to potassium currents with distinct functional properties. Our results provide a molecular basis for retinal dysfunction in patients with mutations in the KCNV2 gene encoding KV8.2. Introduction late the rate and repolarization of action potentials (Liu and Bean, 2014; Kimm et al., 2015). The K 2 subunits can only form func- Voltage-gated potassium channels (KV) are a large and diverse V family of ion channels expressed in neuronal and non-neuronal tional heterotetramers within the KV2 family, or with the so- called “modifier/silent” subunits, K 5, K 6, K 8, and K 9(K S). tissues. The KV2 family (Shab) subunits, KV2.1 and KV2.2, are V V V V V high-voltage–activated and are widely expressed in central neu- KVS subunits cannot form functional homomeric channels but interact with K 2 channels to alter their biophysical properties rons where they generate delayed rectifier KV currents that regu- V (for review, see Bocksteins and Snyders, 2012; Bocksteins, 2016). Received Aug. 25, 2017; revised Jan. 18, 2018; accepted Feb. 11, 2018. The KVS subunits are presumed to produce tissue-specific or Author contributions: D.B.Y., R.A.K., and T.P. edited the paper; T.P. wrote the first draft of the paper; J.G.-P., cell-type-specific modification of KV2 channel gating properties; D.B.Y., and T.P. designed research; J.G.-P., D.B.Y., and T.P. performed research; J.G.-P., D.B.Y., R.A.K., and T.P. however, the localization, subunit interactions, and functional analyzed data; T.P. wrote the paper. significance of K S subunits remain poorly understood. This study was supported by National Institutes of Health Grants EY024265 to T.P., P30 EY010572 and T32 V EY23211toOregonHealth&ScienceUniversity,P51OD011092toOregonNationalPrimateResearchCenter,andP30 KV8.2 is the only silent subunit that has thus far been impli- EY003176toUniversityofCalifornia,Berkeley.WethankDr.RowlandTaylorforhelpfulfeedbackonthemanuscript; cated in human disease. Variants/mutations in the gene that and Drs. John Ng and David Wilson (Oregon Health & Science University) for providing human retinal samples. encodes this protein, KCNV2, have been implicated in epilepsy The authors declare no competing financial interests. susceptibility (Jorge et al., 2011), as well as in an inherited pho- Correspondence should be addressed to Dr. Teresa Puthussery, School of Optometry & Helen Wills Neuroscience Institute, 406 Minor Hall, University of California, Berkeley, Berkeley, CA 94720. E-mail: [email protected]. toreceptor dystrophy known as “cone-dystrophy with supernor- DOI:10.1523/JNEUROSCI.2440-17.2018 mal rod response” (Wu et al., 2006; Wissinger et al., 2008, 2011; Copyright © 2018 the authors 0270-6474/18/383414-14$15.00/0 Zelinger et al., 2013). The photoreceptor dystrophy causes re- • Gayet-Primo et al. KV2/KV8.2 Channels in Primate Photoreceptors J. Neurosci., April 4, 2018 • 38(14):3414–3427 • 3415 duced visual acuity, photophobia, color vision deficits, and versed the dorsal to ventral retina to rule out any staining variations that variable loss of night vision (Gouras et al., 1983). Because KV8.2 might arise due to differences in cone distribution. Mouse eyes were subunits cannot form functional homomeric channels, visual oriented using previously described protocols (Wei et al., 2010). In some experiments with K 8.2 antibodies (ms and rb), sections were treated for dysfunction presumably arises from alterations in KV2/KV8.2 het- V antigen retrieval with 0.15 mg/ml pepsin in 0.2 M HCl at 25°C. The eromeric channels. Indeed, in expression systems, KV2.1/KV8.2 het- eromers exhibit more hyperpolarized activation potentials, show reaction was stopped with washes in ice-cold 0.1 M PB before proceeding less inactivation, and have lower current densities than homo- to the blocking step. Nonspecific binding sites were blocked in 10% normal horse serum, 1% Triton X-100, 0.025% NaN inPBSfor1hat meric K 2.1 channels (Czirja´k et al., 2007; Smith et al., 2012). 3 V 25°C. Primary antibodies were diluted in 3% normal horse serum, 1% Although KV2.1 and KV8.2 genes are expressed in human and Triton X-100, 0.025% NaN3 in PBS and applied to sections overnight at rodent outer retina (Wu et al., 2006; Czirja´k et al., 2007), the 25°C. After washing, sections were incubated in secondary antibodies localization of the proteins remains unclear (Pinto and Klumpp, diluted in 3% normal horse serum, 0.025% NaN3 inPBSfor1hat25°C. 1998; Aslanidis et al., 2014). Moreover, to our knowledge, inter- Secondary antibodies were raised in donkey or goat and conjugated to actions between KV2 and KV8.2 subunits have not yet been dem- AlexaFluor-488, -594, or -647 (Invitrogen) or Cy3 (Jackson ImmunoRe- onstrated in any native tissue. search Laboratories). Isotype selective anti-mouse secondary antibodies Delayed rectifier KV currents are continuously active in photore- were used when mouse monoclonal antibodies of different IgG sub- ceptors. In darkness, photoreceptors are tonically depolarized by a classes were combined in double labeling experiments. In some experi- “dark current” mediated by cGMP-gated cation channels in the ments, we used AlexaFluor-488-conjugated peanut agglutinin (PNA) outer segment. This inward dark current is balanced by a nonin- lectin to localize mouse cones (Invitrogen, L21409). Slides were mounted in Mowiol mounting medium. activating delayed rectifier KV current in the inner segment, dubbed I (Beech and Barnes, 1989; Yan and Matthews, 1992), Confocal images were acquired on an Olympus FV1000 laser scanning Kx ϫ which stabilizes the resting potential at ϳϪ40 mV (Schneeweis microscope (60 /1.4 objective, 488, 559, and 643 nm laser lines) or on a ϫ and Schnapf, 1995). Upon light stimulation, the photoreceptors Zeiss LSM 880 laser scanning microscope (63 /1.4 objective, 488, 561, hyperpolarize and the K current shuts off with some delay, lead- and 594 nm laser lines). Images were acquired sequentially to prevent V fluorescent cross talk. In some cases, adjustments to image brightness and ing to a delayed depolarizing phase that renders the photorecep- contrast were made with ImageJ (Fiji) 1.48r or Adobe Photoshop CC. tor voltage response more transient and bandpass filtered. The For quantification of K 2 staining intensity in human cones, we ac- molecular identity of the channels that mediate the delayed rec- V quired confocal z stacks of vertical sections of retina labeled for KV2.2 and tifier KV current has not been resolved. Here, we demonstrate the S cone-opsin. Optimal PMT gain and laser power settings were deter- differential expression of KV2.1, KV2.2, and KV8.2 in rod and mined while viewing a high-low lookup table to prevent pixel saturation. cone photoreceptors in human and mouse retina, show protein- Acquisition settings were then kept constant for imaging different re- binding interactions between these K channel subunits in the gions from the same sample. Confocal image stacks were analyzed using retina, and provide physiological and pharmacological evidence Fiji. For each cone, we found the central plane of the inner segment and that there are functional Kv2-containing channels in nonhuman then made 10 straight line profile plots (10 pixel width) perpendicular to primate photoreceptors. Our results provide insights into the the membrane. The peaks of the intensity plots were then aligned and molecular basis for visual deficits in patients with KCNV2 averaged together in Igor Pro 6.0 (Wavemetrics) to find the average mutations. membrane intensity for each cone inner segment. Data were analyzed using paired t tests with an ␣ value of 0.05. Immunoblots and immunoprecipitation. For immunoprecipitation and Materials and Methods immunoblotting experiments of macaque retina, tissues were either snap Tissue preparation.
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