Characterization of C-terminal Splice Variants of Cav1.4 Ca2+ Channels in Human Retina.

Voltage-gated Ca(2+) channels (Cav) undergo extensive alternative splicing that greatly enhances their functional diversity in excitable cells. Here, we characterized novel splice variants of the cytoplasmic C-terminal domain of Cav1.4 Ca(2+) channels that regulate neurotransmitter release in photoreceptors in the retina. These variants lack a portion of exon 45 and/or the entire exon 47 (Cav1.4Δex p45, Cav1.4Δex 47, Cav1.4Δex p45,47) and are expressed in the retina of primates but not mice. Although the electrophysiological properties of Cav1.4Δex p45 are similar to those of full-length channels (Cav1.4FL), skipping of exon 47 dramatically alters Cav1.4 function. Deletion of exon 47 removes part of a C-terminal automodulatory domain (CTM) previously shown to suppress Ca(2+)-dependent inactivation (CDI) and to cause a positive shift in the voltage dependence of channel activation. Exon 47 is crucial for these effects of the CTM because variants lacking this exon show intense CDI and activate at more hyperpolarized voltages than Cav1.4FL The robust CDI of Cav1.4Δex 47 is suppressed by CaBP4, a regulator of Cav1.4 channels in photoreceptors. Although CaBP4 enhances activation of Cav1.4FL, Cav1.4Δex 47 shows similar voltage-dependent activation in the presence and absence of CaBP4. We conclude that exon 47 encodes structural determinants that regulate CDI and voltage-dependent activation of Cav1.4, and is necessary for modulation of channel activation by CaBP4.

Characterization of Cav1.4 complexes (α11.4, ß2, and α2δ4) in HEK293T cells and in the retina.

In photoreceptor synaptic terminals, voltage-gated Cav1.4 channels mediate Ca(2+) signals required for transmission of visual stimuli. Like other high voltage-activated Cav channels, Cav1.4 channels are composed of a main pore-forming Cav1.4 α1 subunit and auxiliary ß and α2δ subunits. Of the four distinct classes of ß and α2δ, ß2 and α2δ4 are thought to co-assemble with Cav1.4 α1 subunits in photoreceptors. However, an understanding of the functional properties of this combination of Cav subunits is lacking. Here, we provide evidence that Cav1.4 α1, ß2, and α2δ4 contribute to Cav1.4 channel complexes in the retina and describe their properties in electrophysiological recordings. In addition, we identified a variant of ß2, named here ß2X13, which, along with ß2a, is present in photoreceptor terminals. Cav1.4 α1, ß2, and α2δ4 were coimmunoprecipitated from lysates of transfected HEK293 cells and mouse retina and were found to interact in the outer plexiform layer of the retina containing the photoreceptor synaptic terminals, by proximity ligation assays. In whole-cell patch clamp recordings of transfected HEK293T cells, channels (Cav1.4 α1 + ß2X13) containing α2δ4 exhibited weaker voltage-dependent activation than those with α2δ1. Moreover, compared with channels (Cav1.4 α1 + α2δ4) with ß2a, ß2X13-containing channels exhibited greater voltage-dependent inactivation. The latter effect was specific to Cav1.4 because it was not seen for Cav1.2 channels. Our results provide the first detailed functional analysis of the Cav1.4 subunits that form native photoreceptor Cav1.4 channels and indicate potential heterogeneity in these channels conferred by ß2a and ß2X13 variants.

Structural insights into activation of the retinal L-type Ca²âº channel (Cav1.4) by Ca²âº-binding protein 4 (CaBP4).

CaBP4 modulates Ca(2+)-dependent activity of L-type voltage-gated Ca(2+) channels (Cav1.4) in retinal photoreceptor cells. Mg(2+) binds to the first and third EF-hands (EF1 and EF3), and Ca(2+) binds to EF1, EF3, and EF4 of CaBP4. Here we present NMR structures of CaBP4 in both Mg(2+)-bound and Ca(2+)-bound states and model the CaBP4 structural interaction with Cav1.4. CaBP4 contains an unstructured N-terminal region (residues 1-99) and four EF-hands in two separate lobes. The N-lobe consists of EF1 and EF2 in a closed conformation with either Mg(2+) or Ca(2+) bound at EF1. The C-lobe binds Ca(2+) at EF3 and EF4 and exhibits a Ca(2+)-induced closed-to-open transition like that of calmodulin. Exposed residues in Ca(2+)-bound CaBP4 (Phe(137), Glu(168), Leu(207), Phe(214), Met(251), Phe(264), and Leu(268)) make contacts with the IQ motif in Cav1.4, and the Cav1.4 mutant Y1595E strongly impairs binding to CaBP4. We conclude that CaBP4 forms a collapsed structure around the IQ motif in Cav1.4 that we suggest may promote channel activation by disrupting an interaction between IQ and the inhibitor of Ca(2+)-dependent inactivation domain.

Anti-PD-1 chimeric antigen receptor T cells efficiently target SIV-infected CD4+ T cells in germinal centers.

Programmed cell death protein 1 (PD-1) is an immune checkpoint marker commonly expressed on memory T cells and enriched in latently HIV-infected CD4+ T cells. We engineered an anti-PD-1 chimeric antigen receptor (CAR) to assess the impact of PD-1 depletion on viral reservoirs and rebound dynamics in SIVmac239-infected rhesus macaques (RMs). Adoptive transfer of anti-PD-1 CAR T cells was done in 2 SIV-naive and 4 SIV-infected RMs on antiretroviral therapy (ART). In 3 of 6 RMs, anti-PD-1 CAR T cells expanded and persisted for up to 100 days concomitant with the depletion of PD-1+ memory T cells in blood and tissues, including lymph node CD4+ follicular helper T (TFH) cells. Loss of TFH cells was associated with depletion of detectable SIV RNA from the germinal center (GC). However, following CAR T infusion and ART interruption, there was a marked increase in SIV replication in extrafollicular portions of lymph nodes, a 2-log higher plasma viremia relative to controls, and accelerated disease progression associated with the depletion of CD8+ memory T cells. These data indicate anti-PD-1 CAR T cells depleted PD-1+ T cells, including GC TFH cells, and eradicated SIV from this immunological sanctuary.

A suite of enhancer AAVs and transgenic mouse lines for genetic access to cortical cell types.

The mammalian cortex is comprised of cells classified into types according to shared properties. Defining the contribution of each cell type to the processes guided by the cortex is essential for understanding its function in health and disease. We used transcriptomic and epigenomic cortical cell type taxonomies from mouse and human to define marker genes and putative enhancers and created a large toolkit of transgenic lines and enhancer AAVs for selective targeting of cortical cell populations. We report evaluation of fifteen new transgenic driver lines, two new reporter lines, and >800 different enhancer AAVs covering most subclasses of cortical cells. The tools reported here as well as the scaled process of tool creation and modification enable diverse experimental strategies towards understanding mammalian cortex and brain function.

Novel engineered chimeric engulfment receptors trigger T cell effector functions against SIV-infected CD4+ T cells.

Adoptive therapy with genetically engineered T cells offers potential for infectious disease treatment in immunocompromised persons. HIV/simian immunodeficiency virus (SIV)-infected cells express phosphatidylserine (PS) early post infection. We tested whether chimeric engulfment receptor (CER) T cells designed to recognize PS-expressing cells could eliminate SIV-infected cells. Lentiviral CER constructs composed of the extracellular domain of T cell immunoglobulin and mucin domain containing 4 (TIM-4), the PS receptor, and engulfment signaling domains were transduced into primary rhesus macaque (RM) T cells. We measured PS binding and T cell engulfment of RM CD4+ T cells infected with SIV expressing GFP and in vitro, TIM-4 CER CD4+ T cells effectively killed SIV-infected cells, which was dependent on TIM-4 binding to PS. Enhanced killing of SIV-infected CD4+ T cells by CER and chimeric antigen receptor T cell combinations was also observed. This installation of innate immune functions into T cells presents an opportunity to enhance elimination of SIV-infected cells, and studies to evaluate their effect in vivo are warranted.

Immune inactivation of anti-simian immunodeficiency virus chimeric antigen receptor T cells in rhesus macaques.

Chimeric antigen receptor (CAR) T cell therapies are being investigated as potential HIV cures and designed to target HIV reservoirs. Monoclonal antibodies (mAbs) targeting the simian immunodeficiency virus (SIV) envelope allowed us to investigate the potency of single-chain variable fragment (scFv)-based anti-SIV CAR T cells. In vitro, CAR T cells expressing the scFv to both the variable loop 1 (V1) or V3 of the SIV envelope were highly potent at eliminating SIV-infected T cells. However, in preclinical studies, in vivo infusion of these CAR T cells in rhesus macaques (RMs) resulted in lack of expansion and no detectable in vivo antiviral activity. Injection of envelope-expressing antigen-presenting cells (APCs) 1 week post-CAR T cell infusion also failed to stimulate CAR T cell expansion in vivo. To investigate this in vitro versus in vivo discrepancy, we examined host immune responses directed at CAR T cells. A humoral immune response against the CAR scFv was detected post-infusion of the anti-SIV CAR T cells; anti-SIV IgG antibodies present in plasma of SIV-infected animals were associated with inhibited CAR T cell effector functions. These data indicate that lack of in vivo expansion and efficacy of CAR T cells might be due to antibodies blocking the interaction between the CAR scFv and its epitope.

Real-Time Killing Assays to Assess the Potency of a New Anti-Simian Immunodeficiency Virus Chimeric Antigen Receptor T Cell.

The success of chimeric antigen receptor (CAR) T cell therapies for treating leukemia has resulted in a booming interest for the technology. Expression of a CAR in T cells allows redirection of their natural cytolytic activity toward cells presenting a specific designated surface antigen. Although CAR T cell therapies have thus far shown promising results mostly in B cell malignancy trials, interest in their potential to treat other diseases is on the rise, including using CAR T cells to control human immunodeficiency virus infection. The assessment of CAR T cell potency toward specific targets in vitro is a critical preclinical step. In this study, we describe novel assays that monitor the cytotoxicity of candidate CAR T cells toward simian immunodeficiency virus (SIV) infected CD4 T cells. The assays involve live cell imaging using a fluorescence microscopy system that records in real time the disappearance or appearance of targets infected with SIV carrying a fluorescent protein gene. The assays are highly reproducible, and their rapid turn around and reduced cost present a significant advance regarding the efficient preclinical evaluation of CAR T cell constructs and are broadly applicable to potential human diseases that could benefit from CAR T cell therapy.

Phosphorylation of the Ca2+-binding protein CaBP4 by protein kinase C zeta in photoreceptors.

CaBP4 is a calmodulin-like neuronal calcium-binding protein that is crucial for the development and/or maintenance of the cone and rod photoreceptor synapse. Previously, we showed that CaBP4 directly regulates Ca(v)1 L-type Ca2+ channels, which are essential for normal photoreceptor synaptic transmission. Here, we show that the function of CaBP4 is regulated by phosphorylation. CaBP4 is phosphorylated by protein kinase C zeta (PKCzeta) at serine 37 both in vitro and in the retina and colocalizes with PKCzeta in photoreceptors. CaBP4 phosphorylation is greater in light-adapted than dark-adapted mouse retinas. In electrophysiological recordings of cells transfected with Ca(v)1.3 and CaBP4, mutation of the serine 37 to alanine abolished the effect of CaBP4 in prolonging the Ca2+ current through Ca(v)1.3 channel, whereas inactivating mutations in the CaBP4 Ca2+-binding sites strengthened Ca(v)1.3 modulation. These findings demonstrate how light-stimulated changes in CaBP4 phosphorylation and Ca2+ binding may regulate presynaptic Ca2+ signals in photoreceptors.

Interaction and colocalization of CaBP4 and Unc119 (MRG4) in photoreceptors.

PURPOSE: To characterize the interaction of the neuron-specific protein CaBP4 with the synaptic photoreceptor protein Unc119 homolog (MRG4). METHODS: The interaction of CaBP4 and Unc119 was studied using affinity chromatography, yeast two-hybrid system, coimmunoprecipitation, and gel overlay assay. The colocalization of CaBP4 and Unc119 was analyzed using immunohistochemistry. Unc119, CaBP4, and synaptic proteins were examined in photoreceptors using immunohistochemistry and in synaptic tangential sections of flatmounted frozen retinas using Western blot analysis. RESULTS: Biochemical evidence supported the interaction of CaBP4 with Unc119. CaBP4 and Unc119 colocalized in the photoreceptor synapse of adult retina and during postnatal retinal development. A reduction in Unc119 levels was observed in the photoreceptor terminals of CaBP4-knockout mice compared with wild-type mice and was higher than the reduction of other synaptic proteins. CONCLUSIONS: This study provides evidence for the interaction of CaBP4 with Unc119 at the photoreceptor synapse. This interaction suggests a functional relationship between CaBP4 and Unc119, further supporting a role for these proteins in neurotransmitter release and in the maintenance of the photoreceptor synapse.

Essential role of Ca2+-binding protein 4, a Cav1.4 channel regulator, in photoreceptor synaptic function.

CaBP1-8 are neuronal Ca(2+)-binding proteins with similarity to calmodulin (CaM). Here we show that CaBP4 is specifically expressed in photoreceptors, where it is localized to synaptic terminals. The outer plexiform layer, which contains the photoreceptor synapses with secondary neurons, was thinner in the Cabp4(-/-) mice than in control mice. Cabp4(-/-) retinas also had ectopic synapses originating from rod bipolar and horizontal cells tha HJt extended into the outer nuclear layer. Responses of Cabp4(-/-) rod bipolars were reduced in sensitivity about 100-fold. Electroretinograms (ERGs) indicated a reduction in cone and rod synaptic function. The phenotype of Cabp4(-/-) mice shares similarities with that of incomplete congenital stationary night blindness (CSNB2) patients. CaBP4 directly associated with the C-terminal domain of the Ca(v)1.4 alpha(1)-subunit and shifted the activation of Ca(v)1.4 to hyperpolarized voltages in transfected cells. These observations indicate that CaBP4 is important for normal synaptic function, probably through regulation of Ca(2+) influx and neurotransmitter release in photoreceptor synaptic terminals.

Differential modulation of Ca(v)2.1 channels by calmodulin and Ca2+-binding protein 1.

Ca(v)2.1 channels, which mediate P/Q-type Ca2+ currents, undergo Ca2+/calmodulin (CaM)-dependent inactivation and facilitation that can significantly alter synaptic efficacy. Here we report that the neuronal Ca2+-binding protein 1 (CaBP1) modulates Ca(v)2.1 channels in a manner that is markedly different from modulation by CaM. CaBP1 enhances inactivation, causes a depolarizing shift in the voltage dependence of activation, and does not support Ca2+-dependent facilitation of Ca(v)2.1 channels. These inhibitory effects of CaBP1 do not require Ca2+, but depend on the CaM-binding domain in the alpha1 subunit of Ca(v)2.1 channels (alpha12.1). CaBP1 binds to the CaM-binding domain, co-immunoprecipitates with alpha12.1 from transfected cells and brain extracts, and colocalizes with alpha12.1 in discrete microdomains of neurons in the hippocampus and cerebellum. Our results identify an interaction between Ca2+ channels and CaBP1 that may regulate Ca2+-dependent forms of synaptic plasticity by inhibiting Ca2+ influx into neurons.

Splicing of an automodulatory domain in Cav1.4 Ca2+ channels confers distinct regulation by calmodulin.

Ca2+ influx through Cav1.4 L-type Ca2+ channels supports the sustained release of glutamate from photoreceptor synaptic terminals in darkness, a process that is critical for vision. Consistent with this role, Cav1.4 exhibits weak Ca2+-dependent inactivation (CDI)-a negative feedback regulation mediated by Ca2+-bound calmodulin (CaM). CaM binds to a conserved IQ domain in the proximal C-terminal domain of Cav channels, but in Cav1.4, a C-terminal modulatory domain (CTM) disrupts interactions with CaM. Exon 47 encodes a portion of the CTM and is deleted in a Cav1.4 splice variant (Cav1.4Δex47) that is highly expressed in the human retina. Cav1.4Δex47 exhibits CDI and enhanced voltage-dependent activation, similar to that caused by a mutation that is associated with congenital stationary night blindness type 2, in which the CTM is deleted (K1591X). The presence of CDI and very negative activation thresholds in a naturally occurring variant of Cav1.4 are perplexing considering that these properties are expected to be maladaptive for visual signaling and result in night blindness in the case of K1591X. Here we show that Cav1.4Δex47 and K1591X exhibit fundamental differences in their regulation by CaM. In Cav1.4Δex47, CDI requires both the N-terminal (N lobe) and C-terminal (C lobe) lobes of CaM to bind Ca2+, whereas CDI in K1591X is driven mainly by Ca2+ binding to the C lobe. Moreover, the CaM N lobe causes a Ca2+-dependent enhancement of activation of Cav1.4Δex47 but not K1591X. We conclude that the residual CTM in Cav1.4Δex47 enables a form of CaM N lobe regulation of activation and CDI that is absent in K1591X. Interaction with the N lobe of CaM, which is more sensitive to global elevations in cytosolic Ca2+ than the C lobe, may allow Cav1.4Δex47 to be modulated by a wider range of synaptic Ca2+ concentrations than K1591X; this may distinguish the normal physiological function of Cav1.4Δex47 from the pathological consequences of K1591X.

Lack of CaBP1/Caldendrin or CaBP2 Leads to Altered Ganglion Cell Responses.

Calcium-binding proteins (CaBPs) form a subfamily of calmodulin-like proteins that were cloned from the retina. CaBP4 and CaBP5 have been shown to be important for normal visual function. Although CaBP1/caldendrin and CaBP2 have been shown to modulate various targets in vitro, it is not known whether they contribute to the transmission of light responses through the retina. Therefore, we generated mice that lack CaBP2 or CaBP1/caldendrin (Cabp2-/- and Cabp1-/- ) to test whether these CaBPs are essential for normal retinal function. By immunohistochemistry, the overall morphology of Cabp1-/- and Cabp2-/- retinas and the number of synaptic ribbons appear normal; transmission electron microscopy shows normal tethered ribbon synapses and synaptic vesicles as in wild-type retinas. However, whole-cell patch clamp recordings showed that light responses of retinal ganglion cells of Cabp2-/- and Cabp1-/- mice differ in amplitude and kinetics from those of wild-type mice. We conclude that CaBP1/caldendrin and CaBP2 are not required for normal gross retinal and synapse morphology but are necessary for the proper transmission of light responses through the retina; like other CaBPs, CaBP1/caldendrin and CaBP2 likely act by modulating presynaptic Ca2+-dependent signaling mechanisms.

Expression and Localization of CaBP Ca2+ Binding Proteins in the Mouse Cochlea.

CaBPs are a family of EF-hand Ca2+ binding proteins that are structurally similar to calmodulin. CaBPs can interact with, and yet differentially modulate, effectors that are regulated by calmodulin, such as Cav1 voltage-gated Ca2+ channels. Immunolabeling studies suggest that multiple CaBP family members (CaBP1, 2, 4, and 5) are expressed in the cochlea. To gain insights into the respective auditory functions of these CaBPs, we characterized the expression and cellular localization of CaBPs in the mouse cochlea. By quantitative reverse transcription PCR, we show that CaBP1 and CaBP2 are the major CaBPs expressed in mouse cochlea both before and after hearing onset. Of the three alternatively spliced variants of CaBP1 (caldendrin, CaBP1-L, and CaBP1-S) and CaBP2 (CaBP2-alt, CaBP2-L, CaBP2-S), caldendrin and CaBP2-alt are the most abundant. By in situ hybridization, probes recognizing caldendrin strongly label the spiral ganglion, while probes designed to recognize all three isoforms of CaBP1 weakly label both the inner and outer hair cells as well as the spiral ganglion. Within the spiral ganglion, caldendrin/CaBP1 labeling is associated with cells resembling satellite glial cells. CaBP2-alt is strongly expressed in inner hair cells both before and after hearing onset. Probes designed to recognize all three variants of CaBP2 strongly label inner hair cells before hearing onset and outer hair cells after the onset of hearing. Thus, CaBP1 and CaBP2 may have overlapping roles in regulating Ca2+ signaling in the hair cells, and CaBP1 may have an additional function in the spiral ganglion. Our findings provide a framework for understanding the role of CaBP family members in the auditory periphery.

Ca2+-binding protein-1 facilitates and forms a postsynaptic complex with Cav1.2 (L-type) Ca2+ channels.

Ca2+-binding protein-1 (CaBP1) is a Ca2+-binding protein that is closely related to calmodulin (CaM) and localized in somatodendritic regions of principal neurons throughout the brain, but how CaBP1 participates in postsynaptic Ca2+ signaling is not known. Here, we describe a novel role for CaBP1 in the regulation of Ca2+ influx through Ca(v)1.2 (L-type) Ca2+ channels. CaBP1 interacts directly with the alpha1 subunit of Ca(v)1.2 at sites that also bind CaM. CaBP1 binding to one of these sites, the IQ domain, is Ca2+ dependent and competitive with CaM binding. The physiological significance of this interaction is supported by the association of Ca(v)1.2 and CaBP1 in postsynaptic density fractions purified from rat brain. Moreover, in double-label immunofluorescence experiments, CaBP1 and Ca(v)1.2 colocalize in numerous cell bodies and dendrites of neurons, particularly in pyramidal cells in the CA3 region of the hippocampus and in the dorsal cortex. In electrophysiological recordings of cells transfected with Ca(v)1.2, CaBP1 greatly prolonged Ca2+ currents, prevented Ca2+-dependent inactivation, and caused Ca2+-dependent facilitation of currents evoked by step depolarizations and repetitive stimuli. These effects contrast with those of CaM, which promoted strong Ca2+-dependent inactivation of Ca(v)1.2 with these same voltage protocols. Our findings reveal how Ca2+-binding proteins, such as CaM and CaBP1, differentially adjust Ca2+ influx through Ca(v)1.2 channels, which may specify diverse modes of Ca2+ signaling in neurons.

A critical role of CaBP4 in the cone synapse.

PURPOSE: CaBP4, a photoreceptor-specific protein of the rods and cones, is essential for the development and maintenance of the mouse photoreceptor synapse. In this study, double CaBP4/rod alpha-transducin knockout (Cabp4(-/-)Gnat1(-/-)) mice lacking the rod-mediated component of electrophysiologic responses were generated and analyzed to investigate the role of CaBP4 in cones. METHODS: The retinal morphology and physiologic function of 2-month-old Cabp4(-/-)Gnat1(-/-) mice were analyzed using immunocytochemistry, electron microscopy, and single-flash and flicker electroretinography (ERG). RESULTS: The thickness of the outer plexiform layer and the number of photoreceptor terminals in Cabp4(-/-)Gnat1(-/-) mice were reduced to levels similar to those of Cabp4(-/-) mice. Single-flash and flicker ERG showed that the amplitude and sensitivity of the b-wave in the Cabp4(-/-)Gnat1(-/-) mice were severely attenuated compared with those in wild-type and Gnat1(-/-) mice. CONCLUSIONS: Results indicate that the cone synaptic function in Cabp4(-/-)Gnat1(-/-) mice was severely disrupted, whereas the morphologic defects observed in Cabp4(-/-)Gnat1(-/-) mice were similar to those of single Cabp4(-/-) knockout mice. This and a previous study reveal that CaBP4 is critical for signal transmission from rods and cones to second-order neurons.

Novel targeting strategy for generating mouse models with defects in the retinoid cycle.

In addition to RDH5, other enzymes capable of oxidizing 11-cis-retinol are present within the retinal pigment epithelium, Müller cells and/or photoreceptors. Candidate proteins have meanwhile been identified. To study the physiological and pathological aspects of these enzymes, mice in which these genes are no longer functional are being generated. A fast-targeting strategy for the disruption of genes was developed. Generation of double and triple knockouts will aid in determining if these retinol dehydrogenases are responsible for the remaining 11-cis-retinol oxidation observed in RDH5 knockout animals.

Calmodulin and Ca2+-binding proteins (CaBPs): variations on a theme.

Ca2+ is a ubiquitous second messenger that frequently exerts its effects through Ca2+-binding proteins. In response to changes in the intracellular [Ca2+], Ca2+-binding proteins modulate the cellular activities of enzymes, channels and structural proteins. Multiple Ca2+-binding proteins are expressed in the retina and, in most cases, in a unique cellular and sub-cellular manner. CaBPs are retinal Ca2+-binding proteins displaying a high similarity to calmodulin (CaM). CaBPs are able to mimic some of the interactions of CaM with effector enzymes, although their physiological role has not yet been resolved. CaBPs could be cell-type specific proteins that play a key role in the Ca2+ signaling of specialized retinal neurons.

Protein phosphatase 2A dephosphorylates CaBP4 and regulates CaBP4 function.

PURPOSE: CaBP4 is a neuronal Ca(2+)-binding protein that is expressed in the retina and in the cochlea, and is essential for normal photoreceptor synaptic function. CaBP4 is phosphorylated by protein kinase C zeta (PKCζ) in the retina at serine 37, which affects its interaction with and modulation of voltage-gated Ca(v)1 Ca(2+) channels. In this study, we investigated the potential role and functional significance of protein phosphatase 2A (PP2A) in CaBP4 dephosphorylation. METHODS: The effect of protein phosphatase inhibitors, light, and overexpression of PP2A subunits on CaBP4 dephosphorylation was measured in in vitro assays. Pull-down experiments using retinal or transfected HEK293 cell lysates were used to investigate the association between CaBP4 and PP2A subunits. Electrophysiologic recordings of cotransfected HEK293 cells were performed to analyze the effect of CaBP4 dephosphorylation in modulating Ca(v)1.3 currents. RESULTS: PP2A inhibitors, okadaic acid (OA), and fostriecin, but not PP1 selective inhibitors, NIPP-1, and inhibitor 2, block CaBP4 dephosphorylation in retinal lysates. Increased phosphatase activity in light-dependent conditions reverses phosphorylation of CaBP4 by PKCζ. In HEK293 cells, overexpression of PP2A enhances the rate of dephosphorylation of CaBP4. In addition, inhibition of protein phosphatase activity by OA increases CaBP4 phosphorylation and potentiates the modulatory effect of CaBP4 on Ca(v)1.3 Ca(2+) channels in HEK293T cells. CONCLUSIONS: This study provides evidence that CaBP4 is dephosphorylated by PP2A in the retina. Our findings reveal a novel role for protein phosphatases in regulating CaBP4 function in the retina, which may fine tune presynaptic Ca(2+) signals at the photoreceptor synapse.