Aaron M. Beedle

RIM1 confers sustained activity and neurotransmitter vesicle anchoring to presynaptic Ca2+ channels.

The molecular organization of presynaptic active zones is important for the neurotransmitter release that is triggered by depolarization-induced Ca2+ influx. Here, we demonstrate a previously unknown interaction between two components of the presynaptic active zone, RIM1 and voltage-dependent Ca2+ channels (VDCCs), that controls neurotransmitter release in mammalian neurons. RIM1 associated with VDCC β-subunits via its C terminus to markedly suppress voltage-dependent inactivation among different neuronal VDCCs. Consistently, in pheochromocytoma neuroendocrine PC12 cells, acetylcholine release was significantly potentiated by the full-length and C-terminal RIM1 constructs, but membrane docking of vesicles was enhanced only by the full-length RIM1. The β construct beta-AID dominant negative, which disrupts the RIM1-β association, accelerated the inactivation of native VDCC currents, suppressed vesicle docking and acetylcholine release in PC12 cells, and inhibited glutamate release in cultured cerebellar neurons. Thus, RIM1 association with β in the presynaptic active zone supports release via two distinct mechanisms: sustaining Ca2+ influx through inhibition of channel inactivation, and anchoring neurotransmitter-containing vesicles in the vicinity of VDCCs.

The CACNA1F Gene Encodes an L-Type Calcium Channel with Unique Biophysical Properties and Tissue Distribution

Glutamate release from rod photoreceptors is dependent on a sustained calcium influx through L-type calcium channels. Missense mutations in the CACNA1F gene in patients with incomplete X-linked congenital stationary night blindness implicate the Cav1.4 calcium channel subtype. Here, we describe the functional and pharmacological properties of transiently expressed human Cav1.4 calcium channels. Cav1.4 is shown to encode a dihydropyridine-sensitive calcium channel with unusually slow inactivation kinetics that are not affected by either calcium ions or by coexpression of ancillary calcium channel β subunits. Additionally, the channel supports a large window current and activates near -40 mV in 2 mM external calcium, making Cav1.4 ideally suited for tonic calcium influx at typical photoreceptor resting potentials. Introduction of base pair changes associated with four incomplete X-linked congenital night blindness mutations showed that only the G369D alteration affected channel activation properties. Immunohistochemical analyses show that, in contrast with previous reports, Cav1.4 is widely distributed outside the retina, including in the immune system, thus suggesting a broader role in human physiology.

ORL1 receptor–mediated internalization of N-type calcium channels

The inhibition of N-type calcium channels by opioid receptor like receptor 1 (ORL1) is a key mechanism for controlling the transmission of nociceptive signals. We recently reported that signaling complexes consisting of ORL1 receptors and N-type channels mediate a tonic inhibition of calcium entry. Here we show that prolonged (∼30 min) exposure of ORL1 receptors to their agonist nociceptin triggers an internalization of these signaling complexes into vesicular compartments. This effect is dependent on protein kinase C activation, occurs selectively for N-type channels and cannot be observed with μ-opioid or angiotensin receptors. In expression systems and in rat dorsal root ganglion neurons, the nociceptin-mediated internalization of the channels is accompanied by a significant downregulation of calcium entry, which parallels the selective removal of N-type calcium channels from the plasma membrane. This may provide a new means for long-term regulation of calcium entry in the pain pathway.

Agonist-independent modulation of N-type calcium channels by ORL1 receptors

We have investigated modulation of voltage-gated calcium channels by nociceptin (ORL1) receptors. In rat DRG neurons and in tsA-201 cells, nociceptin mediated a pronounced inhibition of N-type calcium channels, whereas other calcium channel subtypes were unaffected. In tsA-201 cells, expression of N-type channels with human ORL1 resulted in a voltage-dependent G-protein inhibition of the channel that occurred in the absence of nociceptin, the ORL1 receptor agonist. Consistent with this observation, native N-type channels of small nociceptive dorsal root ganglion (DRG) neurons also had tonic inhibition by G proteins. Biochemical characterization showed the existence of an N-type calcium channel–ORL1 receptor signaling complex, which efficiently exposes N-type channels to constitutive ORL1 receptor activity. Calcium channel activity is thus regulated by changes in ORL1 receptor expression, which provides a possible molecular mechanism for the development of tolerance to opioid receptor agonists.

Expression of voltage-gated Ca2+ channel subtypes in cultured astrocytes.

Transient intracellular [Ca2+] increases in astrocytes from influx and/or release from internal stores can release glutamate and thereby modulate synaptic transmission in adjacent neurons. Electrophysiological studies have shown that cultured astrocytes express voltage‐dependent Ca2+ channels but their molecular identities have remained unexplored. We therefore performed RT‐PCR analysis with primers directed to different voltage‐gated Ca2+ channel α1 subunits. In primary cultures of astrocytes, we detected mRNA transcripts for the α1B (N‐type), α1C (L‐type), α1D (L‐type), α1E (R‐type), and α1G (T‐type), but not α1A (P/Q‐type), voltage‐gated Ca2+ channels. We then used antibodies against all of the Ca2+ channel subunits to confirm protein expression, via Western blots, and localization by means of immunocytochemistry. In Western blot analysis, we observed immunoreactive bands corresponding to the appropriate α1 subunit proteins. Western blots showed an expression pattern similar to PCR results in that we detected proteins for the α1B (N‐type), α1C (L‐type), α1D (L‐type), α1E (R‐type), and α1G (T‐type), but not α1A (P/Q‐type). Using immunocytochemistry, we observed Ca2+ channel expression for these subunits in punctate clusters on plasmamembrane of GFAP‐expressing astrocytes. These results confirm that cultured astrocytes express corresponding proteins to several high‐ and low‐threshold Ca2+ channels but not α1A (P/Q‐type). Overall, our data indicate that astrocytes express multiple types of voltage‐gated Ca2+ channels, hinting at a complex regulation of Ca2+ homeostasis in glial cells. GLIA 41:347–353, 2003.

Inhibition of transiently expressed low- and high-voltage-activated calcium channels by trivalent metal cations.

Calcium channels are important regulators of neuronal excitability and contribute to transmitter release, calcium dependent gene expression, and oscillatory behavior in many cell types. Under physiological conditions, native low-voltage (T-type)- and high-voltage-activated (HVA) currents are potently inhibited by trivalent cations. However, the presence of multiple calcium channel isoforms has hampered our ability to unequivocally assess the effects of trivalent cations on channel activity. Here, we describe the actions of nine trivalent metal ions on transiently expressed a1G (Cav3.1) T-type calcium channels cloned from human brain. In 2 mM external barium solution, yttrium most potently inhibited a1G current (IC50 = 28 nM), followed by erbium > gadolinium ~ cerium > holmium > ytterbium > neodymium > lanthanum » scandium. With the exception of scandium, blocking affinity was loosely correlated with decreasing ionic radius. A detailed characterization of yttrium block revealed a 25-fold decrease in blocking affinity when the external concentration of charge carrier was increased from 2 mM to 20 mM. In 20 mM barium, yttrium also effectively inhibited various types of cloned HVA channels indicating that this ion is a nonselective blocker. For all calcium channels examined, yttrium preferentially inhibited inward over outward current, but block was otherwise voltage independent. In addition to peak current inhibition, P/Q- and L-type channels underwent a unique speeding of the macroscopic time course of inactivation. Whereas peak current block of a1A channels was highly sensitive to the external charge carrier concentration, the inactivation effects mediated by yttrium were not, suggesting that the two effects are due to distinct mechanisms. Moreover, the speeding effect was greatly attenuated by manipulations that slowed the inactivation kinetics of the channels. Thus, our evidence suggests that yttrium effects are mediated by two distinct events: peak current block likely occurring by occlusion of the pore, and kinetic speeding arising from yttrium interactions with the channel that alter the state of the inactivation gate.n

Expression of T-type calcium channel splice variants in human glioma

In humans, three isoforms of the T‐type (Cav3.1) calcium‐channel α1 subunit have been reported as a result of alternate splicing of exons 25 and 26 in the III–IV linker region (Cav3.1a, Cav3.1b or Cav3.1bc). In the present study, we report that human glioma express Cav3.1 channels in situ, that splicing of these exons is uniquely regulated and that there is expression of a glioma‐specific novel T‐type variant (Cav3.1ac). Seven human glioma samples were collected at surgery, RNA was extracted, and cDNA was produced for RT‐PCR analysis. In addition, three glioma cell lines (U87, U563, and U251N), primary cultures of human fetal astrocytes, as well as adult and fetal human brain cDNA were used. Previously described Cav3.1 splice variants were present in glioma samples, cultured cells and whole brain. Consistent with the literature, our results reveal that in the normal adult brain, Cav3.1a transcripts predominate, while Cav3.1b is mostly fetal‐specific. RT‐PCR results on glioma and glioma cell lines showed that Cav3.1 expression in tumor cells resemble fetal brain expression pattern as Cav3.1bc is predominantly expressed. In addition, we identified a novel splice variant, Cav3.1ac, expressed in three glioma biopsies and one glioma cell line, but not in normal brain or fetal astrocytes. Transient expression of this variant demonstrates that Cav3.1ac displays similar current‐voltage and steady‐state inactivation properties compared with Cav3.1b, but a slower recovery from inactivation. Taken together, our data suggest glioma‐specific Cav3.1 gene regulation, which could possibly contribute to tumor pathogenesis.

Evidence for a role of dystroglycan regulating the membrane architecture of astroglial endfeet

The dystrophin–dystroglycan complex (DDC) is a molecular array of proteins in muscle and brain cells. The central component of the DDC is dystroglycan, which comprises α‐ and β‐subunits. α‐Dystroglycan (α‐DG) binds to extracellular matrix components such as agrin, whereas β‐dystroglycan (β‐DG) is a membrane‐spanning protein linking α‐DG to the cytoskeleton and other intracellular components such as α‐syntrophin. In astrocytes, α‐syntrophin binds to the water channel protein aquaporin‐4 (AQP4). Recently, it has been shown that AQP4 expression is unaltered in agrin‐knockout mice, but that formation of orthogonal arrays of particles (OAPs), consisting of AQP4, is abnormal. As the brain‐selective deletion of the DG gene causes a disorganization of the astroglial endfeet, we investigated whether DG deletion has an impact on AQP4. Western blotting revealed reduced AQP4 in the parenchymal but not in the superficial compartment of the astrocyte‐conditioned DG‐knockout mouse brain. Accordingly, immunohistochemical stainings of AQP4 revealed a selective loss of AQP4 in perivascular but not in superficial astroglial endfeet. In both superficial and perivascular endfeet of the DG‐knockout brain, we observed a loss of OAPs. We conclude that in the absence of DG the majority of superficial AQP4 molecules did not form OAPs, and that expression of AQP4 in perivascular endfeet is compromised. However, the decreased number of perivascular AQP4 molecules obviously did form a few OAPs, even in the absence of DG.

Fukutin-related protein associates with the sarcolemmal dystrophin-glycoprotein complex

Mutations in fukutin-related protein (FKRP) give rise to mild and more severe forms of muscular dystrophy. FKRP patients have reduced glycosylation of the extracellular protein dystroglycan, and FKRP itself shows sequence similarity to glycosyltransferases, implicating FKRP in the processing of dystroglycan. However, FKRP localization is controversial, and no FKRP complexes are known, so any FKRP-dystroglycan link remains elusive. Here, we demonstrate a novel FKRP localization in vivo; in mouse, both endogenous and recombinant FKRP are present at the sarcolemma. Biochemical analyses revealed that mouse muscle FKRP and dystroglycan co-enrich and co-fractionate, indicating that FKRP coexists with dystroglycan in the native dystrophin-glycoprotein complex. Furthermore, FKRP sedimentation shifts with dystroglycan in disease models involving the dystrophin-glycoprotein complex, and sarcolemmal FKRP immunofluorescence mirrors that of dystroglycan in muscular dystrophy mice, suggesting that FKRP localization may be mediated by dystroglycan. These data offer the first evidence of an FKRP complex in muscle and suggest that FKRP may influence the glycosylation status of dystroglycan from within the sarcolemmal dystrophin-glycoprotein complex.

Mouse fukutin deletion impairs dystroglycan processing and recapitulates muscular dystrophy

Dystroglycan is a transmembrane glycoprotein that links the extracellular basement membrane to cytoplasmic dystrophin. Disruption of the extensive carbohydrate structure normally present on α-dystroglycan causes an array of congenital and limb girdle muscular dystrophies known as dystroglycanopathies. The essential role of dystroglycan in development has hampered elucidation of the mechanisms underlying dystroglycanopathies. Here, we developed a dystroglycanopathy mouse model using inducible or muscle-specific promoters to conditionally disrupt fukutin (Fktn), a gene required for dystroglycan processing. In conditional Fktn-KO mice, we observed a near absence of functionally glycosylated dystroglycan within 18 days of gene deletion. Twenty-week-old KO mice showed clear dystrophic histopathology and a defect in glycosylation near the dystroglycan O-mannose phosphate, whether onset of Fktn excision driven by muscle-specific promoters occurred at E8 or E17. However, the earlier gene deletion resulted in more severe phenotypes, with a faster onset of damage and weakness, reduced weight and viability, and regenerating fibers of smaller size. The dependence of phenotype severity on the developmental timing of muscle Fktn deletion supports a role for dystroglycan in muscle development or differentiation. Moreover, given that this conditional Fktn-KO mouse allows the generation of tissue- and timing-specific defects in dystroglycan glycosylation, avoids embryonic lethality, and produces a phenotype resembling patient pathology, it is a promising new model for the study of secondary dystroglycanopathy.