PRISM :: Browsing by Author "Zamponi, Gerald W"

Browsing by Author "Zamponi, Gerald W"

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Open Access
A CACNA1A variant associated with trigeminal neuralgia alters the gating of Cav2.1 channels
(2021-01-07) Gambeta, Eder; Gandini, Maria A; Souza, Ivana A; Ferron, Laurent; Zamponi, Gerald W
Abstract A novel missense mutation in the CACNA1A gene that encodes the pore forming α1 subunit of the CaV2.1 voltage-gated calcium channel was identified in a patient with trigeminal neuralgia. This mutation leads to a substitution of proline 2455 by histidine (P2455H) in the distal C-terminus region of the channel. Due to the well characterized role of this channel in neurotransmitter release, our aim was to characterize the biophysical properties of the P2455H variant in heterologously expressed CaV2.1 channels. Whole-cell patch clamp recordings of wild type and mutant CaV2.1 channels expressed in tsA-201 cells reveal that the mutation mediates a depolarizing shift in the voltage-dependence of activation and inactivation. Moreover, the P2455H mutant strongly reduced calcium-dependent inactivation of the channel that is consistent with an overall gain of function. Hence, the P2455H CaV2.1 missense mutation alters the gating properties of the channel, suggesting that associated changes in CaV2.1-dependent synaptic communication in the trigeminal system may contribute to the development of trigeminal neuralgia.
Open Access
A rare CACNA1H variant associated with amyotrophic lateral sclerosis causes complete loss of Cav3.2 T-type channel activity
(2020-03-06) Stringer, Robin N; Jurkovicova-Tarabova, Bohumila; Huang, Sun; Haji-Ghassemi, Omid; Idoux, Romane; Liashenko, Anna; Souza, Ivana A; Rzhepetskyy, Yuriy; Lacinova, Lubica; Van Petegem, Filip; Zamponi, Gerald W; Pamphlett, Roger; Weiss, Norbert
Abstract Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder characterized by the progressive loss of cortical, brain stem and spinal motor neurons that leads to muscle weakness and death. A previous study implicated CACNA1H encoding for Cav3.2 calcium channels as a susceptibility gene in ALS. In the present study, two heterozygous CACNA1H variants were identified by whole genome sequencing in a small cohort of ALS patients. These variants were functionally characterized using patch clamp electrophysiology, biochemistry assays, and molecular modeling. A previously unreported c.454GTAC > G variant produced an inframe deletion of a highly conserved isoleucine residue in Cav3.2 (p.ΔI153) and caused a complete loss-of-function of the channel, with an additional dominant-negative effect on the wild-type channel when expressed in trans. In contrast, the c.3629C > T variant caused a missense substitution of a proline with a leucine (p.P1210L) and produced a comparatively mild alteration of Cav3.2 channel activity. The newly identified ΔI153 variant is the first to be reported to cause a complete loss of Cav3.2 channel function. These findings add to the notion that loss-of-function of Cav3.2 channels associated with rare CACNA1H variants may be risk factors in the complex etiology of ALS.
Open Access
A T-type channel-calmodulin complex triggers αCaMKII activation
(2017-08-11) Asmara, Hadhimulya; Micu, Ileana; Rizwan, Arsalan P; Sahu, Giriraj; Simms, Brett A; Zhang, Fang-Xiong; Engbers, Jordan D T; Stys, Peter K; Zamponi, Gerald W; Turner, Ray W
Abstract Calmodulin (CaM) is an important signaling molecule that regulates a vast array of cellular functions by activating second messengers involved in cell function and plasticity. Low voltage-activated calcium channels of the Cav3 family have the important role of mediating low threshold calcium influx, but were not believed to interact with CaM. We find a constitutive association between CaM and the Cav3.1 channel at rest that is lost through an activity-dependent and Cav3.1 calcium-dependent CaM dissociation. Moreover, Cav3 calcium influx is sufficient to activate αCaMKII in the cytoplasm in a manner that depends on an intact Cav3.1 C-terminus needed to support the CaM interaction. Our findings thus establish that T-type channel calcium influx invokes a novel dynamic interaction between CaM and Cav3.1 channels to trigger a signaling cascade that leads to αCaMKII activation.
Open Access
Analgesic effect of a mixed T-type channel inhibitor/CB2 receptor agonist
(BioMed Central, 2013-07-01) Gadotti, Vinicius M; You, Haitao; Petrov, Ravil R; Berger, N Daniel; Diaz, Philippe; Zamponi, Gerald W
Open Access
Analgesic effects of optogenetic inhibition of basolateral amygdala inputs into the prefrontal cortex in nerve injured female mice
(2019-12-04) Gadotti, Vinicius M; Zhang, Zizhen; Huang, Junting; Zamponi, Gerald W
Abstract Peripheral nerve injury can lead to remodeling of brain circuits, and this can cause chronification of pain. We have recently reported that male mice subjected to spared injury of the sciatic nerve undergo changes in the function of the medial prefrontal cortex (mPFC) that culminate in reduced output of layer 5 pyramidal cells. More recently, we have shown that this is mediated by alterations in synaptic inputs from the basolateral amygdala (BLA) into GABAergic interneurons in the mPFC. Optogenetic inhibition of these inputs reversed mechanical allodynia and thermal hyperalgesia in male mice. It is known that the processing of pain signals can exhibit marked sex differences. We therefore tested whether the dysregulation of BLA to mPFC signaling is equally altered in female mice. Injection of AAV-Arch3.0 constructs into the BLA followed by implantation of a fiberoptic cannula into the mPFC in sham and SNI operated female mice was carried out, and pain behavioral responses were measured in response to yellow light mediated activation of this inhibitory opsin. Our data reveal that Arch3.0 activation leads to a marked increase in paw withdrawal thresholds and latencies in response to mechanical and thermal stimuli, respectively. However, we did not observe nerve injury-induced changes in mPFC layer 5 pyramidal cell output in female mice. Hence, the observed light-induced analgesic effects may be due to compensation for dysregulated neuronal circuits downstream of the mPFC.
Open Access
Ankyrin B and Ankyrin B variants differentially modulate intracellular and surface Cav2.1 levels
(2019-09-02) Choi, Catherine S W; Souza, Ivana A; Sanchez-Arias, Juan C; Zamponi, Gerald W; Arbour, Laura T; Swayne, Leigh A
Abstract Ankyrin B (AnkB) is an adaptor and scaffold for motor proteins and various ion channels that is ubiquitously expressed, including in the brain. AnkB has been associated with neurological disorders such as epilepsy and autism spectrum disorder, but understanding of the underlying mechanisms is limited. Cav2.1, the pore-forming subunit of P/Q type voltage gated calcium channels, is a known interactor of AnkB and plays a crucial role in neuronal function. Here we report that wildtype AnkB increased overall Cav2.1 levels without impacting surface Cav2.1 levels in HEK293T cells. An AnkB variant, p.S646F, which we recently discovered to be associated with seizures, further increased overall Cav2.1 levels, again with no impact on surface Cav2.1 levels. AnkB p.Q879R, on the other hand, increased surface Cav2.1 levels in the presence of accessory subunits α2δ1 and β4. Additionally, AnkB p.E1458G decreased surface Cav2.1 irrespective of the presence of accessory subunits. In addition, we found that partial deletion of AnkB in cortex resulted in a decrease in overall Cav2.1 levels, with no change to the levels of Cav2.1 detected in synaptosome fractions. Our work suggests that depending on the particular variant, AnkB regulates intracellular and surface Cav2.1. Notably, expression of the AnkB variant associated with seizure (AnkB p.S646F) caused further increase in intracellular Cav2.1 levels above that of even wildtype AnkB. These novel findings have important implications for understanding the role of AnkB and Cav2.1 in the regulation of neuronal function in health and disease.
Open Access
Anxiolytic effects of the flavonoid luteolin in a mouse model of acute colitis
(2019-12-26) Gadotti, Vinicius M; Zamponi, Gerald W
Abstract Anxiety related disorders commonly occur in association with major depressive disorder (MDD) in individuals suffering from peripheral inflammation, with a higher prevalence among IBS patients. We have previously shown that the bioflavonoid luteolin has pronounced analgesic and antidepressant-like effects in mice with dextran sodium sulfate (DSS)-induced colitis. Here, we further evaluate the biological effect of luteolin as a possible anxiolytic agent in DSS treated mice. Anxiolytic action was evaluated using the open field test (OF), the novelty suppressed feeding test (NSFT) and the elevated plus maze test (EPM). Luteolin increased the number of crossings in the center of the OF apparatus, reduced the latency to interact with the food pellet in the NSFT, and increased the time spent in the open arms in the EPM. These results suggest luteolin as a possible natural anxiolytic molecule without sedative effects, thus reinforcing its therapeutic potential for the comorbidities involving peripheral inflammation, pain, mood and anxiety-related disorders.
Open Access
Cav3.2 calcium channel interactions with the epithelial sodium channel ENaC
(2019-02-08) Garcia-Caballero, Agustin; Gandini, Maria A; Huang, Shuo; Chen, Lina; Souza, Ivana A; Dang, Yan L; Stutts, M. J; Zamponi, Gerald W
Abstract This study describes the functional interaction between Cav3.2 calcium channels and the Epithelial Sodium Channel (ENaC). β-ENaC subunits showed overlapping expression with endogenous Cav3.2 calcium channels in the thalamus and hypothalamus as detected by immunostaining. Moreover, β- and γ-ENaC subunits could be co-immunoprecipitated with Cav3.2 calcium channels from brain lysates, dorsal horn and lumbar dorsal root ganglia. Mutation of a cluster of lysines present in the intracellular N-terminus region of β-ENaC (K4R/ K5R/ K9R/ K16R/ K23R) reduced interactions with Cav3.2 calcium channels. Αβγ-ENaC channels enhanced Cav3.2 calcium channel trafficking to the plasma membrane in tsA-201 cells. This effect was reciprocal such that Cav3.2 channel expression also enhanced β-ENaC trafficking to the cell surface. T-type current density was increased when fully assembled αβγ-ENaC channels were transiently expressed in CAD cells, a neuronal derived cell line. Altogether, these findings reveal ENaC as an interactor and potential regulator of Cav3.2 calcium channels expressed in neuronal tissues.
Open Access
Cav3.2 T-type calcium channels control acute itch in mice
(2020-09-01) Gadotti, Vinicius M; Kreitinger, Joanna M; Wageling, Nicholas B; Budke, Dylan; Diaz, Philippe; Zamponi, Gerald W
Abstract Cav3.2 T-type calcium channels are important mediators of nociceptive signaling, but their roles in the transmission of itch remains poorly understood. Here we report a key involvement of these channels as key modulators of itch/pruritus-related behavior. We compared scratching behavior responses between wild type and Cav3.2 null mice in models of histamine- or chloroquine-induced itch. We also evaluated the effect of the T-type calcium channel blocker DX332 in male and female wild-type mice injected with either histamine or chloroquine. Cav3.2 null mice exhibited decreased scratching responses during both histamine- and chloroquine-induced acute itch. DX332 co-injected with the pruritogens inhibited scratching responses of male and female mice treated with either histamine or chloroquine. Altogether, our data provide strong evidence that Cav3.2 T-type channels exert an important role in modulating histamine-dependent and -independent itch transmission in the primary sensory afferent pathway, and highlight these channels as potential pharmacological targets to treat pruritus.
Open Access
Differential modulation of NMDA and AMPA receptors by cellular prion protein and copper ions
(2018-10-25) Huang, Sun; Chen, Lina; Bladen, Chris; Stys, Peter K; Zamponi, Gerald W
Abstract N-Methyl-D-aspartate receptors (NMDARs) and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs) are two major types of ionotropic glutamate receptors involved in synaptic transmission. However, excessive activity of these receptors can be cytotoxic and thus their function must be precisely controlled. We have previously reported that NMDA receptor activity is dysregulated following genetic knockout of cellular prion protein (PrPC), and that PrPC regulation of NMDA receptors is copper-dependent. Here, we employed electrophysiological methods to study NMDAR and AMPAR currents of cultured hippocampal neurons from PrPC overexpresser mice. We show that NMDA receptor current amplitude and kinetics are differentially modulated by overexpression of human or mouse PrPC. By contrast, AMPA receptor activity was unaffected. Nonetheless, AMPA receptor activity was modulated by copper ions in a manner similar to what we previously reported for NMDA receptors. Taken together, our findings reveal that AMPA and NMDA receptors are differentially regulated by PrPC, but share common modulation by copper ions.
Open Access
Differential regulation of Cav2.2 channel exon 37 variants by alternatively spliced μ-opioid receptors
(2019-11-27) Gandini, Maria A; Souza, Ivana A; Raval, Dvij; Xu, Jin; Pan, Ying-Xian; Zamponi, Gerald W
Abstract We have examined the regulation of mutually exclusive Cav2.2 exon 37a and b variants by the mouse μ-opioid receptor (mMOR) C-terminal splice variants 1, 1C and 1O in tsA-201 cells. Electrophysiological analyses revealed that both channel isoforms exhibit DAMGO-induced voltage-dependent (Gβγ-mediated) inhibition and its recovery by voltage pre-pulses, as well as a voltage-independent component. However, the two channel isoforms differ in their relative extent of voltage-dependent and independent inhibition, with Cav2.2-37b showing significantly more voltage-dependent inhibition upon activation of the three mMOR receptors studied. In addition, coexpression of either mMOR1 or mMOR1C results in an agonist-independent reduction in the peak current density of Cav2.2-37a channels, whereas the peak current density of Cav2.2-37b does not appear to be affected. Interestingly, this decrease is not due to an effect on channel expression at the plasma membrane, as demonstrated by biotinylation experiments. We further examined the mechanism underlying the agonist-independent modulation of Cav2.2-37a by mMOR1C. Incubation of cells with pertussis toxin did not affect the mMOR1C mediated inhibition of Cav2.2-37a currents, indicating a lack of involvement of Gi/o signaling. However, when a Src tyrosine kinase inhibitor was applied, the effect of mMOR1C was lost. Moreover, when we recorded currents using a Cav2.2-37a mutant in which tyrosine 1747 was replaced with phenylalanine (Y1747F), the agonist independent effects of mMOR1C were abolished. Altogether our findings show that Cav2.2-37a and Cav2.2-37b isoforms are subject to differential regulation by C-terminal splice variants of mMORs, and that constitutive mMOR1C activity and downstream tyrosine kinase activity exert a selective inhibition of the Cav2.2-37a splice variant, an N-type channel isoform that is highly enriched in nociceptors. Our study provides new insights into the roles of the MOR full-length C-terminal variants in modulating Cav2.2 channel isoform activities.
Open Access
Disrupting USP5/Cav3.2 interactions protects female mice from mechanical hypersensitivity during peripheral inflammation
(2018-10-19) Gadotti, Vinicius M; Zamponi, Gerald W
Abstract Cav3.2 T-type calcium channels are important for the signaling of nociceptive information in the primary afferent pain pathway. During neuropathy and peripheral inflammation, Cav3.2 channels are upregulated due to an increased association with the deubiquitinase USP5. Disrupting these interactions in male mice by the use of cell permeant peptides reverses mechanical and thermal hypersensitivity. Here we explore the effects of interfering with USP5 binding to the channel in female mice with synchronized estrous cycle. We show that intrathecal delivery of a cell-penetrating TAT peptide corresponding to the UBPc domain of USP5 fully reverses mechanical hypersensitivity in mice intraplantarly injected with Complete Freund’s Adjuvant. Hence, the USP5 mediated dysregulation of Cav3.2 channel activity does not exhibit sex differences, and potential therapeutics targeting this interaction should be effective in both male and female subjects.
Open Access
Functional identification of potential non-canonical N-glycosylation sites within Cav3.2 T-type calcium channels
(2020-11-11) Ficelova, Vendula; Souza, Ivana A; Cmarko, Leos; Gandini, Maria A; Stringer, Robin N; Zamponi, Gerald W; Weiss, Norbert
Abstract Low-voltage-activated T-type calcium channels are important contributors to nervous system function. Post-translational modification of these channels has emerged as an important mechanism to control channel activity. Previous studies have documented the importance of asparagine (N)-linked glycosylation and identified several asparagine residues within the canonical consensus sequence N-X-S/T that is essential for the expression and function of Cav3.2 channels. Here, we explored the functional role of non-canonical N-glycosylation motifs in the conformation N-X-C based on site directed mutagenesis. Using a combination of electrophysiological recordings and surface biotinylation assays, we show that asparagines N345 and N1780 located in the motifs NVC and NPC, respectively, are essential for the expression of the human Cav3.2 channel in the plasma membrane. Therefore, these newly identified asparagine residues within non-canonical motifs add to those previously reported in canonical sites and suggest that N-glycosylation of Cav3.2 may also occur at non-canonical motifs to control expression of the channel in the plasma membrane. It is also the first study to report the functional importance of non-canonical N-glycosylation motifs in an ion channel.
Open Access
Identification of a molecular gating determinant within the carboxy terminal region of Cav3.3 T-type channels
(2019-04-08) Jurkovicova-Tarabova, Bohumila; Cmarko, Leos; Rehak, Renata; Zamponi, Gerald W; Lacinova, Lubica; Weiss, Norbert
Abstract The physiological functions controlled by T-type channels are intrinsically dependent on their gating properties, and alteration of T-type channel activity is linked to several human disorders. Therefore, it is essential to develop a clear understanding of the structural determinants responsible for the unique gating features of T-type channels. Here, we have investigated the specific role of the carboxy terminal region by creating a series a deletion constructs expressed in tsA-201 cells and analyzing them by patch clamp electrophysiology. Our data reveal that the proximal region of the carboxy terminus contains a structural determinant essential for shaping several gating aspects of Cav3.3 channels, including voltage-dependence of activation and inactivation, inactivation kinetics, and coupling between the voltage sensing and the pore opening of the channel. Altogether, our data are consistent with a model in which the carboxy terminus stabilizes the channel in a closed state.
Open Access
Interactions of Rabconnectin-3 with Cav2 calcium channels
(2019-06-28) Gandini, Maria A; Souza, Ivana A; Fan, Jing; Li, Katherine; Wang, Decheng; Zamponi, Gerald W
Abstract This study describes the interaction between Cav2 calcium channels and Rabconnectin-3, a di-subunit protein that is associated with synaptic vesicles. Immunostaining reveals that both Rabconnectin-3α (RB-3α) and Rabconnectin-3β (RB-3β) are colocalized in mouse hippocampal neurons. Co-immunoprecipitations from brain tissue is consistent with the formation of a protein complex between RB-3α and RB-3β and both Cav2.2 and the related Cav2.1 calcium channel. The coexpression of either RB-3α or RB-3β with Cav2.2 calcium channels in tsA-201 cells led to a reduction in Cav2.2 current density without any effects on the voltage-dependence of activation or inactivation. Coexpression of both Rabconnectin-3 subunits did not cause an additive effect on current densities. Finally, the presence of Rabconnectin-3 did not interfere with μ-opioid receptor mediated Gβγ modulation of Cav2.2 channels. Altogether, our findings show that Rabconnectin-3 has the propensity to regulate calcium entry mediated by Cav2.2 channels.
Open Access
Pathogenic Cav3.2 channel mutation in a child with primary generalized epilepsy
(2019-10-24) Souza, Ivana A; Gandini, Maria A; Zhang, Fang-Xiong; Mitchell, Wendy G; Matsumoto, Joyce; Lerner, Jason; Pierson, Tyler M; Zamponi, Gerald W
Abstract Two paternally-inherited missense variants in CACNA1H were identified and characterized in a 6-year-old child with generalized epilepsy. Febrile and unprovoked seizures were present in this child. Both variants were expressed in cis or isolation using human recombinant Cav3.2 calcium channels in tsA-201 cells. Whole-cell patch-clamp recordings indicated that one variant (c.3844C > T; p.R1282W) caused a significant increase in current density consistent with a pathogenic gain-of-function phenotype; while the other cis-related variant (c.5294C > T; p.A1765V) had a benign profile.
Open Access
Peripheral nerve injury-induced alterations in VTA neuron firing properties
(2019-11-04) Huang, Shuo; Borgland, Stephanie L; Zamponi, Gerald W
Abstract The ventral tegmental area (VTA) is one of the main brain regions harboring dopaminergic (DA) neurons, and plays important roles in reinforcement and motivation. Recent studies have indicated that DA neurons not only respond to rewarding stimuli, but also to noxious stimuli. Furthermore, VTA DA neurons undergo plasticity during chronic pain. Lateral and medial VTA neurons project to different brain areas, and have been characterized via their distinct electrophysiological properties. In this study, we characterized electrophysiological properties of lateral and medial VTA DA neurons using DAT-cre reporter mice, and examined their plasticity during neuropathic pain states. We observed various DA subpopulations in both the lateral and medial VTA, as defined by action potential firing patterns, independently of synaptic inputs. Our results demonstrated that lateral and medial VTA DA neurons undergo differential plasticity after peripheral nerve injury that leads to neuropathic pain. However, these changes only reside in specific DA subpopulations. This study suggests that lateral and medial VTA DA neurons are differentially affected during neuropathic pain conditions, and emphasizes the importance of subpopulation specificity when targeting VTA DA neurons for treatment of neuropathic pain.
Open Access
Rare functional missense variants in CACNA1H: What can we learn from Writer’s cramp?
(2021-01-21) Huang, Miaozhen; Nibbeling, Esther A R; Lagrand, Tjerk J; Souza, Ivana A; Groen, Justus L; Gandini, Maria A; Zhang, Fang-Xiong; Koelman, Johannes H T M; Adir, Noam; Sinke, Richard J; Zamponi, Gerald W; Tijssen, Marina A J; Verbeek, Dineke S
Abstract Writer’s cramp (WC) is a task-specific focal dystonia that occurs selectively in the hand and arm during writing. Previous studies have shown a role for genetics in the pathology of task-specific focal dystonia. However, to date, no causal gene has been reported for task-specific focal dystonia, including WC. In this study, we investigated the genetic background of a large Dutch family with autosomal dominant‒inherited WC that was negative for mutations in known dystonia genes. Whole exome sequencing identified 4 rare variants of unknown significance that segregated in the family. One candidate gene was selected for follow-up, Calcium Voltage-Gated Channel Subunit Alpha1 H, CACNA1H, due to its links with the known dystonia gene Potassium Channel Tetramerization Domain Containing 17, KCTD17, and with paroxysmal movement disorders. Targeted resequencing of CACNA1H in 82 WC cases identified another rare, putative damaging variant in a familial WC case that did not segregate. Using structural modelling and functional studies in vitro, we show that both the segregating p.Arg481Cys variant and the non-segregating p.Glu1881Lys variant very likely cause structural changes to the Cav3.2 protein and lead to similar gains of function, as seen in an accelerated recovery from inactivation. Both mutant channels are thus available for re-activation earlier, which may lead to an increase in intracellular calcium and increased neuronal excitability. Overall, we conclude that rare functional variants in CACNA1H need to be interpreted very carefully, and additional studies are needed to prove that the p.Arg481Cys variant is the cause of WC in the large Dutch family.
Open Access
SUMOylation regulates USP5-Cav3.2 calcium channel interactions
(2019-08-27) Garcia-Caballero, Agustin; Zhang, Fang-Xiong; Chen, Lina; M’Dahoma, Said; Huang, Junting; Zamponi, Gerald W
Abstract Cav3.2 calcium channels play a key role in nociceptive signaling in the primary afferent pain pathway. We have previously reported the regulation of Cav3.2 calcium channels by the deubiquitinase USP5 and its importance for regulating peripheral transmission of pain signals. Here we describe the regulation of the Cav3.2-USP5 interaction by SUMOylation. We show that endogenous USP5 protein expressed in dorsal root ganglia undergoes SUMOylation, and the level of USP5 SUMOylation is reduced following peripheral nerve injury. SUMO prediction software identified several putative lysines that have the propensity to be targets for SUMO conjugation. A series of single lysine substitutions in an mCherry tagged USP5 construct followed by expression in tsA-201 cells identified lysine K113 as a key target for USP5 SUMO2/3 modification. Finally, Cav3.2 calcium channel immunoprecipitates revealed a stronger interaction of Cav3.2 with a SUMO2/3 resistant USP5-K113R mutant, indicating that SUMO2/3 modification of USP5 reduces its affinity for the calcium channel Cav3.2. Collectively, our data suggest that dysregulation of USP5 SUMOylation after peripheral nerve injury may contribute to the well described alteration in Cav3.2 channel activity during neuropathic pain states.
Open Access
T-type calcium channels functionally interact with spectrin (α/β) and ankyrin B
(2018-05-02) Garcia-Caballero, Agustin; Zhang, Fang-Xiong; Hodgkinson, Victoria; Huang, Junting; Chen, Lina; Souza, Ivana A; Cain, Stuart; Kass, Jennifer; Alles, Sascha; Snutch, Terrance P; Zamponi, Gerald W
Abstract This study describes the functional interaction between the Cav3.1 and Cav3.2 T-type calcium channels and cytoskeletal spectrin (α/β) and ankyrin B proteins. The interactions were identified utilizing a proteomic approach to identify proteins that interact with a conserved negatively charged cytosolic region present in the carboxy-terminus of T-type calcium channels. Deletion of this stretch of amino acids decreased binding of Cav3.1 and Cav3.2 calcium channels to spectrin (α/β) and ankyrin B and notably also reduced T-type whole cell current densities in expression systems. Furthermore, fluorescence recovery after photobleaching analysis of mutant channels lacking the proximal C-terminus region revealed reduced recovery of both Cav3.1 and Cav3.2 mutant channels in hippocampal neurons. Knockdown of spectrin α and ankyrin B decreased the density of endogenous Cav3.2 in hippocampal neurons. These findings reveal spectrin (α/β) / ankyrin B cytoskeletal and signaling proteins as key regulators of T-type calcium channels expressed in the nervous system.