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Alexandra Koschak - One of the best experts on this subject based on the ideXlab platform.

  • Cav1.4 dysfunction and congenital stationary night blindness type 2
    Pflügers Archiv: European Journal of Physiology, 2021
    Co-Authors: Alexandra Koschak, Monica L Fernandezquintero, Thomas Heigl, Marco Ruzza, Hartwig Seitter, Lucia Zanetti
    Abstract:

    Cav1.4 L-type Ca2+ channels are predominantly expressed in retinal neurons, particularly at the photoreceptor terminals where they mediate sustained Ca2+ entry needed for continuous neurotransmitter release at their ribbon synapses. Cav1.4 channel gating properties are controlled by accessory subunits, associated regulatory proteins, and also alternative splicing. In humans, mutations in the CACNA1F gene encoding for Cav1.4 channels are associated with X-linked retinal disorders such as congenital stationary night blindness type 2. Mutations in the Cav1.4 protein result in a spectrum of altered functional channel activity. Several mouse models broadened our understanding of the role of Cav1.4 channels not only as Ca2+ source at retinal synapses but also as synaptic organizers. In this review, we highlight different structural and functional phenotypes of Cav1.4 mutations that might also occur in patients with congenital stationary night blindness type 2. A further important yet mostly neglected aspect that we discuss is the influence of alternative splicing on channel dysfunction. We conclude that currently available functional phenotyping strategies should be refined and summarize potential specific therapeutic options for patients carrying Cav1.4 mutations. Importantly, the development of new therapeutic approaches will permit a deeper understanding of not only the disease pathophysiology but also the physiological function of Cav1.4 channels in the retina.

  • function of cone and cone related pathways in Cav1.4 it mice
    Scientific Reports, 2021
    Co-Authors: Lucia Zanetti, Hartwig Seitter, Irem Kilicarslan, Michael Netzer, Norbert Babai, Alexandra Koschak
    Abstract:

    Cav1.4 L-type calcium channels are predominantly expressed in photoreceptor terminals playing a crucial role for synaptic transmission and, consequently, for vision. Human mutations in the encoding gene are associated with congenital stationary night blindness type-2. Besides rod-driven scotopic vision also cone-driven photopic responses are severely affected in patients. The present study therefore examined functional and morphological changes in cones and cone-related pathways in mice carrying the Cav1.4 gain-of function mutation I756T (Cav1.4-IT) using multielectrode array, patch-clamp and immunohistochemical analyses. Cav1.4-IT ganglion cell responses to photopic stimuli were seen only in a small fraction of cells indicative of a major impairment in the cone pathway. Though cone photoreceptors underwent morphological rearrangements, they retained their ability to release glutamate. Our functional data suggested a postsynaptic cone bipolar cell defect, supported by the fact that the majority of cone bipolar cells showed sprouting, while horizontal cells maintained contacts with cones and cone-to-horizontal cell input was preserved. Furthermore a reduction of basal Ca2+ influx by a calcium channel blocker was not sufficient to rescue synaptic transmission deficits caused by the Cav1.4-IT mutation. Long term treatments with low-dose Ca2+ channel blockers might however be beneficial reducing Ca2+ toxicity without major effects on ganglion cells responses.

  • the physiology pathology and pharmacology of voltage gated calcium channels and their future therapeutic potential
    Pharmacological Reviews, 2015
    Co-Authors: Gerald W Zamponi, Joerg Striessnig, Alexandra Koschak, Annette C Dolphin
    Abstract:

    Voltage-gated calcium channels are required for many key functions in the body. In this review, the different subtypes of voltage-gated calcium channels are described and their physiologic roles and pharmacology are outlined. We describe the current uses of drugs interacting with the different calcium channel subtypes and subunits, as well as specific areas in which there is strong potential for future drug development. Current therapeutic agents include drugs targeting L-type CaV1.2 calcium channels, particularly 1,4-dihydropyridines, which are widely used in the treatment of hypertension. T-type (CaV3) channels are a target of ethosuximide, widely used in absence epilepsy. The auxiliary subunit α2δ-1 is the therapeutic target of the gabapentinoid drugs, which are of value in certain epilepsies and chronic neuropathic pain. The limited use of intrathecal ziconotide, a peptide blocker of N-type (CaV2.2) calcium channels, as a treatment of intractable pain, gives an indication that these channels represent excellent drug targets for various pain conditions. We describe how selectivity for different subtypes of calcium channels (e.g., CaV1.2 and CaV1.3 L-type channels) may be achieved in the future by exploiting differences between channel isoforms in terms of sequence and biophysical properties, variation in splicing in different target tissues, and differences in the properties of the target tissues themselves in terms of membrane potential or firing frequency. Thus, use-dependent blockers of the different isoforms could selectively block calcium channels in particular pathologies, such as nociceptive neurons in pain states or in epileptic brain circuits. Of important future potential are selective CaV1.3 blockers for neuropsychiatric diseases, neuroprotection in Parkinson’s disease, and resistant hypertension. In addition, selective or nonselective T-type channel blockers are considered potential therapeutic targets in epilepsy, pain, obesity, sleep, and anxiety. Use-dependent N-type calcium channel blockers are likely to be of therapeutic use in chronic pain conditions. Thus, more selective calcium channel blockers hold promise for therapeutic intervention.

  • gain of function nature of Cav1.4 l type calcium channels alters firing properties of mouse retinal ganglion cells
    Channels, 2015
    Co-Authors: Dagmar Knoflach, Martin Glosmann, Klaus Schicker, Alexandra Koschak
    Abstract:

    Proper function of Cav1.4 L-type calcium channels is crucial for neurotransmitter release in the retina. Our understanding about how different levels of Cav1.4 channel activity affect retinal function is still limited. In the gain-of-function mouse model Cav1.4-IT we expected a reduction in the photoreceptor dynamic range but still transmission toward retinal ganglion cells. A fraction of Cav1.4-IT ganglion cells responded to light stimulation in multielectrode array recordings from whole-mounted retinas, but showed a significantly delayed response onset. Another significant number of cells showed higher activity in darkness. In addition to structural remodeling observed at the first retinal synapse of Cav1.4-IT mice the functional data suggested a loss of contrast enhancement, a fundamental feature of our visual system. In fact, Cav1.4-IT mouse retinas showed a decline in spatial response and changes in their contrast sensitivity profile. Photoreceptor degeneration was obvious from the nodular structure of cone axons and enlarged pedicles which partly moved toward the outer nuclear layer. Loss of photoreceptors was also expressed as reduced expression of proteins involved in chemical and electrical transmission, as such metabotropic glutamate receptor mGluR6 and the gap junction protein Connexin 36. Such gross changes in retinal structure and function could also explain the diminished visual performance of CSNB2 patients. The expression pattern of the plasma-membrane calcium ATPase 1 which participates in the maintenance of the intracellular calcium homeostasis in photoreceptors was changed in Cav1.4-IT mice. This might be part of a protection mechanism against increased calcium influx, as this is suggested for Cav1.4-IT channels.

  • biophysical characterization of Cav1.4 l type calcium channel mutants causing congenital stationary night blindness type 2 in humans
    BMC Clinical Pharmacology, 2012
    Co-Authors: Klaus Schicker, Verena Burtscher, Dagmar Knoflach, Anamika Singh, Thomas Stockner, Alexandra Koschak
    Abstract:

    Background Cav1.4 L-type calcium channels show unique biophysical properties such as slow inactivation due to the lack of calcium-dependent inactivation (CDI). These properties make Cav1.4 channels appropriate candidates for triggering persistent glutamate release at retinal photoreceptor cell synapses. Mutations in the CACNA1F gene encoding for the Cav1.4 a1 subunit are described in patients with X-linked congenital stationary night blindness type 2 (CSNB2). Impaired transmission between rod photoreceptor cells and second-order neurons manifests as night blindness and various other visual symptoms in the affected individuals.

Amy S Lee - One of the best experts on this subject based on the ideXlab platform.

  • functional impact of a congenital stationary night blindness type 2 mutation depends on subunit composition of Cav1.4 ca2 channels
    Journal of Biological Chemistry, 2020
    Co-Authors: Brittany Williams, Josue A Lopez, Wesley J Maddox, Amy S Lee
    Abstract:

    Voltage-gated Cav1 and Cav2 Ca2+ channels are comprised of a pore-forming α1 subunit (Cav1.1-1.4, Cav2.1-2.3) and auxiliary β (β1-4) and α2δ (α2δ-1-4) subunits. The properties of these channels vary with distinct combinations of Cav subunits and alternative splicing of the encoding transcripts. Therefore, the impact of disease-causing mutations affecting these channels may depend on the identities of Cav subunits and splice variants. Here, we analyzed the effects of a congenital stationary night blindness type 2 (CSNB2)-causing mutation, I745T (IT), in Cav1.4 channels typical of those in human retina: Cav1.4 splice variants with or without exon 47 (Cav1.4+ex47 and Cav1.4Δex47, respectively), and the auxiliary subunits, β2X13 and α2δ-4. We find that IT caused both Cav1.4 splice variants to activate at significantly more negative voltages and with slower deactivation kinetics than the corresponding WT channels. These effects of the IT mutation, along with unexpected alterations in ion selectivity, were generally larger in channels lacking exon 47. The weaker ion selectivity caused by IT led to hyperpolarizing shifts in the reversal potential and large outward currents that were evident in channels containing the auxiliary subunits β2X13 and α2δ-4 but not in those with β2A and α2δ-1. We conclude that the IT mutation stabilizes channel opening and alters ion selectivity of Cav1.4 in a manner that is strengthened by exclusion of exon 47 and inclusion of β2X13 and α2δ-4. Our results reveal complex actions of IT in modifying the properties of Cav1.4 channels, which may influence the pathological consequences of this mutation in retinal photoreceptors.

  • rescue of rod synapses by induction of cav alpha 1f in the mature Cav1.4 knock out mouse retina
    Investigative Ophthalmology & Visual Science, 2019
    Co-Authors: Joseph G Laird, Vasily Kerov, Amy S Lee, Sarah H Gardner, Ariel J Kopel, Sheila A Baker
    Abstract:

    Purpose Cav1.4 is a voltage-gated calcium channel clustered at the presynaptic active zones of photoreceptors. Cav1.4 functions in communication by mediating the Ca2+ influx that triggers neurotransmitter release. It also aids in development since rod ribbon synapses do not form in Cav1.4 knock-out mice. Here we used a rescue strategy to investigate the ability of Cav1.4 to trigger synaptogenesis in both immature and mature mouse rods. Methods In vivo electroporation was used to transiently express Cav α1F or tamoxifen-inducible Cav α1F in a subset of Cav1.4 knock-out mouse rods. Synaptogenesis was assayed using morphologic markers and a vision-guided water maze. Results We found that introduction of Cav α1F to knock-out terminals rescued synaptic development as indicated by PSD-95 expression and elongated ribbons. When expression of Cav α1F was induced in mature animals, we again found restoration of PSD-95 and elongated ribbons. However, the induced expression of Cav α1F led to diffuse distribution of Cav α1F in the terminal instead of being clustered beneath the ribbon. Approximately a quarter of treated animals passed the water maze test, suggesting the rescue of retinal signaling in these mice. Conclusions These data confirm that Cav α1F expression is necessary for rod synaptic terminal development and demonstrate that rescue is robust even in adult animals with late stages of synaptic disease. The degree of rod synaptic plasticity seen here should be sufficient to support future vision-restoring treatments such as gene or cell replacement that will require photoreceptor synaptic rewiring.

  • splicing of an automodulatory domain in Cav1.4 ca2 channels confers distinct regulation by calmodulin
    The Journal of General Physiology, 2018
    Co-Authors: Brittany Williams, Francoise Haeseleer, Amy S Lee
    Abstract:

    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.

  • characterization of c terminal splice variants of Cav1.4 ca2 channels in human retina
    Journal of Biological Chemistry, 2016
    Co-Authors: Francoise Haeseleer, Brittany Williams, Amy S Lee
    Abstract:

    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 β2 and α2δ4 in hek293t cells and in the retina
    Journal of Biological Chemistry, 2015
    Co-Authors: Amy S Lee, Shiyi Wang, Brittany Williams, Jussara Hagen, Todd E Scheetz, Francoise Haeseleer
    Abstract:

    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 β and α2δ subunits. Of the four distinct classes of β and α2δ, β2 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, β2, 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 β2, named here β2X13, which, along with β2a, is present in photoreceptor terminals. Cav1.4 α1, β2, 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 + β2X13) containing α2δ4 exhibited weaker voltage-dependent activation than those with α2δ1. Moreover, compared with channels (Cav1.4 α1 + α2δ4) with β2a, β2X13-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 β2a and β2X13 variants.

Brittany Williams - One of the best experts on this subject based on the ideXlab platform.

  • functional impact of a congenital stationary night blindness type 2 mutation depends on subunit composition of Cav1.4 ca2 channels
    Journal of Biological Chemistry, 2020
    Co-Authors: Brittany Williams, Josue A Lopez, Wesley J Maddox, Amy S Lee
    Abstract:

    Voltage-gated Cav1 and Cav2 Ca2+ channels are comprised of a pore-forming α1 subunit (Cav1.1-1.4, Cav2.1-2.3) and auxiliary β (β1-4) and α2δ (α2δ-1-4) subunits. The properties of these channels vary with distinct combinations of Cav subunits and alternative splicing of the encoding transcripts. Therefore, the impact of disease-causing mutations affecting these channels may depend on the identities of Cav subunits and splice variants. Here, we analyzed the effects of a congenital stationary night blindness type 2 (CSNB2)-causing mutation, I745T (IT), in Cav1.4 channels typical of those in human retina: Cav1.4 splice variants with or without exon 47 (Cav1.4+ex47 and Cav1.4Δex47, respectively), and the auxiliary subunits, β2X13 and α2δ-4. We find that IT caused both Cav1.4 splice variants to activate at significantly more negative voltages and with slower deactivation kinetics than the corresponding WT channels. These effects of the IT mutation, along with unexpected alterations in ion selectivity, were generally larger in channels lacking exon 47. The weaker ion selectivity caused by IT led to hyperpolarizing shifts in the reversal potential and large outward currents that were evident in channels containing the auxiliary subunits β2X13 and α2δ-4 but not in those with β2A and α2δ-1. We conclude that the IT mutation stabilizes channel opening and alters ion selectivity of Cav1.4 in a manner that is strengthened by exclusion of exon 47 and inclusion of β2X13 and α2δ-4. Our results reveal complex actions of IT in modifying the properties of Cav1.4 channels, which may influence the pathological consequences of this mutation in retinal photoreceptors.

  • a dual role for Cav1.4 ca2 channels in the molecular and structural organization of the rod photoreceptor synapse
    eLife, 2020
    Co-Authors: Wesley J Maddox, Vasily Kerov, Sheila A Baker, Brittany Williams, Jussara Hagen, Kate L Randall, Ravi P Yadav, Paul J Derr, Luca Della Santina, Nikolai O Artemyev
    Abstract:

    Synapses are fundamental information processing units that rely on voltage-gated Ca2+ (Cav) channels to trigger Ca2+-dependent neurotransmitter release. Cav channels also play Ca2+-independent roles in other biological contexts, but whether they do so in axon terminals is unknown. Here, we addressed this unknown with respect to the requirement for Cav1.4 L-type channels for the formation of rod photoreceptor synapses in the retina. Using a mouse strain expressing a non-conducting mutant form of Cav1.4, we report that the Cav1.4 protein, but not its Ca2+ conductance, is required for the molecular assembly of rod synapses; however, Cav1.4 Ca2+ signals are needed for the appropriate recruitment of postsynaptic partners. Our results support a model in which presynaptic Cav channels serve both as organizers of synaptic building blocks and as sources of Ca2+ ions in building the first synapse of the visual pathway and perhaps more broadly in the nervous system.

  • splicing of an automodulatory domain in Cav1.4 ca2 channels confers distinct regulation by calmodulin
    The Journal of General Physiology, 2018
    Co-Authors: Brittany Williams, Francoise Haeseleer, Amy S Lee
    Abstract:

    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.

  • characterization of c terminal splice variants of Cav1.4 ca2 channels in human retina
    Journal of Biological Chemistry, 2016
    Co-Authors: Francoise Haeseleer, Brittany Williams, Amy S Lee
    Abstract:

    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 β2 and α2δ4 in hek293t cells and in the retina
    Journal of Biological Chemistry, 2015
    Co-Authors: Amy S Lee, Shiyi Wang, Brittany Williams, Jussara Hagen, Todd E Scheetz, Francoise Haeseleer
    Abstract:

    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 β and α2δ subunits. Of the four distinct classes of β and α2δ, β2 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, β2, 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 β2, named here β2X13, which, along with β2a, is present in photoreceptor terminals. Cav1.4 α1, β2, 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 + β2X13) containing α2δ4 exhibited weaker voltage-dependent activation than those with α2δ1. Moreover, compared with channels (Cav1.4 α1 + α2δ4) with β2a, β2X13-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 β2a and β2X13 variants.

Francoise Haeseleer - One of the best experts on this subject based on the ideXlab platform.

  • splicing of an automodulatory domain in Cav1.4 ca2 channels confers distinct regulation by calmodulin
    The Journal of General Physiology, 2018
    Co-Authors: Brittany Williams, Francoise Haeseleer, Amy S Lee
    Abstract:

    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.

  • characterization of c terminal splice variants of Cav1.4 ca2 channels in human retina
    Journal of Biological Chemistry, 2016
    Co-Authors: Francoise Haeseleer, Brittany Williams, Amy S Lee
    Abstract:

    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 β2 and α2δ4 in hek293t cells and in the retina
    Journal of Biological Chemistry, 2015
    Co-Authors: Amy S Lee, Shiyi Wang, Brittany Williams, Jussara Hagen, Todd E Scheetz, Francoise Haeseleer
    Abstract:

    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 β and α2δ subunits. Of the four distinct classes of β and α2δ, β2 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, β2, 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 β2, named here β2X13, which, along with β2a, is present in photoreceptor terminals. Cav1.4 α1, β2, 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 + β2X13) containing α2δ4 exhibited weaker voltage-dependent activation than those with α2δ1. Moreover, compared with channels (Cav1.4 α1 + α2δ4) with β2a, β2X13-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 β2a and β2X13 variants.

  • structural insights into activation of the retinal l type ca2 channel Cav1.4 by ca2 binding protein 4 cabp4
    Journal of Biological Chemistry, 2014
    Co-Authors: Saebomi Park, Francoise Haeseleer, Krzysztof Palczewski, James B Ames
    Abstract:

    CaBP4 modulates Ca2+-dependent activity of L-type voltage-gated Ca2+ channels (Cav1.4) in retinal photoreceptor cells. Mg2+ binds to the first and third EF-hands (EF1 and EF3), and Ca2+ binds to EF1, EF3, and EF4 of CaBP4. Here we present NMR structures of CaBP4 in both Mg2+-bound and Ca2+-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 Mg2+ or Ca2+ bound at EF1. The C-lobe binds Ca2+ at EF3 and EF4 and exhibits a Ca2+-induced closed-to-open transition like that of calmodulin. Exposed residues in Ca2+-bound CaBP4 (Phe137, Glu168, Leu207, Phe214, Met251, Phe264, and Leu268) 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 Ca2+-dependent inactivation domain.

  • dysregulation of Cav1.4 channels disrupts the maturation of photoreceptor synaptic ribbons in congenital stationary night blindness type 2
    Channels, 2013
    Co-Authors: Vasily Kerov, Francoise Haeseleer, Anurima Majumder, Nikolai O Artemyev, Sheila A Baker
    Abstract:

    Mutations in the gene encoding Cav1.4, CACNA1F, are associated with visual disorders including X-linked incomplete congenital stationary night blindness type 2 (CSNB2). In mice lacking Cav1.4 channels, there are defects in the development of “ribbon” synapses formed between photoreceptors (PRs) and second-order neurons. However, many CSNB2 mutations disrupt the function rather than expression of Cav1.4 channels. Whether defects in PR synapse development due to altered Cav1.4 function are common features contributing to the pathogenesis of CSNB2 is unknown. To resolve this issue, we profiled changes in the subcellular distribution of Cav1.4 channels and synapse morphology during development in wild-type (WT) mice and mouse models of CSNB2. Using Cav1.4-selective antibodies, we found that Cav1.4 channels associate with ribbon precursors early in development and are concentrated at both rod and cone PR synapses in the mature retina. In mouse models of CSNB2 in which the voltage-dependence of Cav1.4 activatio...

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  • protein kinase a modulation of Cav1.4 calcium channels
    Nature Communications, 2016
    Co-Authors: Lingjie Sang, Ivy E Dick, David T Yue
    Abstract:

    The regulation of L-type Ca(2+) channels by protein kinase A (PKA) represents a crucial element within cardiac, skeletal muscle and neurological systems. Although much work has been done to understand this regulation in cardiac CaV1.2 Ca(2+) channels, relatively little is known about the closely related Cav1.4 L-type Ca(2+) channels, which feature prominently in the visual system. Here we find that Cav1.4 channels are indeed modulated by PKA phosphorylation within the inhibitor of Ca(2+)-dependent inactivation (ICDI) motif. Phosphorylation of this region promotes the occupancy of calmodulin on the channel, thus increasing channel open probability (PO) and Ca(2+)-dependent inactivation. Although this interaction seems specific to Cav1.4 channels, introduction of ICDI1.4 to CaV1.3 or CaV1.2 channels endows these channels with a form of PKA modulation, previously unobserved in heterologous systems. Thus, this mechanism may not only play an important role in the visual system but may be generalizable across the L-type channel family.

  • live cell biochemistry implicates protein kinase a modulation of l type Cav1.4 channels
    Biophysical Journal, 2015
    Co-Authors: Lingjie Sang, Ivy E Dick, David T Yue
    Abstract:

    The regulation of L-type Ca2+ channels by protein kinase A (PKA), though biologically crucial, has long remained mechanistically storied and complex, as studied in native cells at one extreme, and through in vitro biochemistry at the other. Here, we adopt a different tactic and focus initially on an intermediate context, using ideas drawn from synthetic biology and live-cell biochemistry. We set out to create a form of PKA modulation in L-type channels, based on our recent findings that: (a) calmodulin (CaM) competes for binding at a channel C-terminal ‘IQ’ domain with an ‘ICDI’ module in the C-terminal extremity of L-type CaV1.3/1.4 channels, and (b) dislodging CaM profoundly suppresses peak channel opening by severalfold and eliminates their Ca2+-dependent inactivation (CDI) (Adams et al (2014), Cell in press). We reasoned that implanting a synthetic phosphorylation site in ICDI might weaken IQ interaction in a PKA-sensitive manner, allowing channels to rebind CaM and undergo CDI. Cognizant that full-bore PKA signaling is best conserved within certain native rather than model cells, we performed live-cell FRET interaction assays (IQ versus ICDI) in adult guinea-pig ventricular myocytes renown for strong PKA signaling. To our surprise in control experiments, we discovered that IQ interaction with the wild-type ICDI of L-type Cav1.4 channels is already sharply attenuated by PKA activation, whereas ICDI modules from other L-type isoforms showed no such modulability. Accordingly, we synthesized chimeric L-type CaV1.3 channels fused to the Cav1.4 ICDI module, and endowed such channels with robust forskolin-dependent enhancement of CDI, as observed in HEK293 cells. For wild-type Cav1.4 channels, we now also resolved analogous forskolin activatable CDI. This effect, discovered through a synthetic live-cell biochemical approach, might underlie the dopaminergic regulation of Cav1.4 implicated in circadian control within the retina.

  • the distal carboxy tail dct of Cav1.4 modulates more than ca2 cam dependent inactivation cdi
    Biophysical Journal, 2012
    Co-Authors: Paul J Adams, Manu B Johny, Ivy E Dick, Hojjat Bazzazi, Phil S Yang, David T Yue
    Abstract:

    Cav1.4 L-type Ca2+ channels populate retinal ribbon synapses, and mediate tonic neurotransmitter release. Mutations in the CACNA1F gene encoding Cav1.4 are implicated in congenital stationary night blindness (CSNB2). The K1591X CSNB2 mutation yields a premature stop that deletes the DCT of Cav1.4. Interestingly, the DCT contains a module that competes with Ca2+-free calmodulin (apoCaM) for binding at the IQ-domain of Cav1.4, thereby tuning channel affinity for apoCaM (Nature 463:968). Since only channels ‘charged’ with apoCaM exhibit Ca2+/CaM-mediated inactivation (CDI), wildtype Cav1.4 channels exhibit little or no CDI, while deleting the DCT yields a resurgence of CDI. The latter effect in K1591X channels has suggested that pathogenesis involves abnormally diminished Ca2+ influx with resulting impairment of photoreceptor signaling. Here, however, we observe a dramatic and unrecognized effect of DCT deletion. Single-channel recordings indicate that wild-type Cav1.4 feature diminutive open probability Po (A. Top, exemplar single-channel trace during voltage ramp. Bottom, Po-V relation averaged over multiple patches). By contrast, K1591X channels exhibit strikingly enhanced Po (B). Hence, this form of CSNB2 likely involves an unexpected Ca2+ overload phenomenon, raising the possibility of therapeutics involving Cav1.4 channel blockade.View Large Image | View Hi-Res Image | Download PowerPoint Slide

  • molecular events beyond apocam preassociation in the cam regulation of cav1 3 channels
    Biophysical Journal, 2011
    Co-Authors: Manu B Johny, Philemon S Yang, David T Yue
    Abstract:

    CaV channel regulation by calmodulin (CaM) is a central prototype for ion-channel modulation. Despite long study, relatively little is known mechanistically, beyond the initial preassociation of Ca2+-free CaM (apoCaM) with an IQ domain on the carboxy-terminus of channels. Most studies have focused on the IQ domain and immediate upstream ‘preIQ’ regions, despite hints that further upstream elements in the carboxy-terminus could be important. Accordingly, we here undertake alanine scanning mutagenesis of the entire carboxy-terminus upstream of the IQ domain (the proximal CI-region, PCI). For analysis, we chose CaV1.3 channels (highly homologous to classic CaV1.2), because they exhibit robust CaM-mediated inactivation (CDI), with particularly well-resolved profiles for both N- and C-lobe forms of inactivation. Several unexpected results were obtained. First, mutations throughout the preIQ domain left CDI essentially unchanged, at odds with functional hotspots in the homologous region of CaV1.2. Second, newly identified segments, situated upstream of the preIQ region, proved selectively critical for the C-lobe form of CDI. Specifically, we argue that the PCI region is the Ca2+/CaM effector site for C-lobe CDI, as revealed by quantitative comparison of the effect of PCI mutations on CDI, to their effect on PCI binding with Ca2+/CaM (‘Ψ-analysis’). Third, we further exploit Ψ-analysis to extend and confirm that the Ca2+/CaM effector site for the N-lobe form of CDI is structurally distinct, residing in the NSCaTE element of the channel amino terminus (Nature 451:830). Finally, while the IQ-domain is a primary site for apoCaM preassociation, our scan surprisingly reveals that PCI harbors additional preassociation sites, especially important for the N-lobe of apoCaM. Overall, this alanine scan of the CaV1.3, together with that of the IQ domain (companion abstract), outlines the long-sought molecular events beyond the initial apoCaM preassociation with the channel.

  • Cav1.4 c tail segment icdi inhibits cav channel inactivation by competing with calmodulin resolution by holochannels and calmodulin fret sensors
    Biophysical Journal, 2009
    Co-Authors: Xiaodong Liu, Philemon S Yang, Wanjun Yang, David T Yue
    Abstract:

    An intriguing variation on calmodulin/CaV channel inactivation (CDI) is the action of a C-tail segment from Cav1.4 channels (ICDI) to eliminate CDI. Introducing ICDI into CaV1.2 or CaV1.3 channels nearly abolishes strong baseline CDI, and a like effect is observed when ICDI is present within Cav1.4 itself. In retina, the effect in Cav1.4 helps sustain Ca2+ influx despite maintained depolarization. Contrasting with clear-cut function, the underlying ICDI mechanism remains controversial. One group proposes that ICDI allosterically inhibits CDI (Wahl-Schott et al PNAS 2006), while another suggests direct competition between calmodulin and ICDI for the channel (Singh et al Nature Neurosci 2006). The discussion hinges on differing calmodulin versus channel peptide assays. Here, we perform functional interaction assays using holochannels within live cells. As baseline, we electrophysiologically characterized CaV1.3 channels fused to an ICDI-containing segment (α1D-ABI-F). These α1D-ABI-F channels exhibited little CDI compared to wild-type CaV1.3. Critically, variations in the ambient calmodulin concentration would only affect competitive versus allosteric mechanisms. Indeed, when calmodulin was depleted by a ‘calmodulin sponge,’ residual CDI in α1D-ABI-F, was totally eliminated. More telling, when calmodulin was over-expressed with α1D-ABI-F, we observed a resurgence of CDI to wild-type CaV1.3 levels. To test for precise agreement with a competitive mechanism, we co-expressed α1D-ABI-F channels with BSCaMIQ, a FRET biosensor of calmodulin (Black et al Biochemistry 2006). Accordingly, both CDI and calmodulin concentrations could be measured within single cells; and pooling data from cells exhibiting variable calmodulin levels permitted explicit resolution of an in situ calmodulin binding curve, in strict agreement with a competitive mechanism. In all, ICDI suppresses CDI by competing with calmodulin for the channel, raising the possibility that natural variations in calmodulin might customize CDI through this mechanism.