Jawed Hamid

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.

Ca(V)3 T-type calcium channel isoforms differentially distribute to somatic and dendritic compartments in rat central neurons.

Spike output in many neuronal cell types is affected by low‐voltage‐activated T‐type calcium currents arising from the Cav3.1, Cav3.2 and Cav3.3 channel subtypes and their splice isoforms. The contributions of T‐type current to cell output is often proposed to reflect a differential distribution of channels to somatic and dendritic compartments, but the subcellular distribution of the various rat T‐type channel isoforms has not been fully determined. We used subtype‐specific Cav3 polyclonal antibodies to determine their distribution in key regions of adult Sprague–Dawley rat brain thought to exhibit T‐type channel expression, and in particular, dendritic low‐voltage‐activated responses. We found a selective subcellular distribution of Cav3 channel proteins in cell types of the neocortex and hippocampus, thalamus, and cerebellar input and output neurons. In general, the Cav3.1 T‐type channel immunolabel is prominent in the soma/proximal dendritic region and Cav3.2 immunolabel in the soma and proximal‐mid dendrites. Cav3.3 channels are distinct in distributing to the soma and over extended lengths of the dendritic arbor of particular cell types. Cav3 distribution overlaps with cell types previously established to exhibit rebound burst discharge as well as those not recognized for this activity. Additional immunolabel in the region of the nucleus in particular cell types was verified as corresponding to Cav3 antigen through analysis of isolated protein fractions. These results provide evidence that different Cav3 channel isoforms may contribute to low‐voltage‐activated calcium‐dependent responses at the somatic and dendritic level, and the potential for T‐type calcium channels to contribute to multiple aspects of neuronal activity.

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.

Fast Inactivation of Voltage-dependent Calcium Channels A HINGED-LID MECHANISM?

We recently described domains II and III as important determinants of fast, voltage-dependent inactivation of R-type calcium channels (Spaetgens, R. L., and Zamponi, G. W. (1999) J. Biol. Chem. 274, 22428–22438). Here we examine in greater detail the structural determinants of inactivation using a series of chimeras comprising various regions of wild type α1C and α1Ecalcium channels. Substitution of the II S6 and/or III S6 segments of α1E into the α1C backbone resulted in rapid inactivation rates that closely approximated those of wild type α1E channels. However, neither individual or combined substitution of the II S6 and III S6 segments could account for the 60 mV more negative half-inactivation potential seen with wild type α1E channels, indicating that the S6 regions contribute only partially to the voltage dependence of inactivation. Interestingly, the converse replacement of α1E S6 segments of domains II, III, or II+III with those of α1Cwas insufficient to significantly slow inactivation rates. Only when the I-II linker region and the domain II and III S6 regions of α1E were concomitantly replaced with α1Csequence could inactivation be abolished. Conversely, introduction of the α1E domain I-II linker sequence into α1C conferred α1E-like inactivation rates, indicating that the domain I-II linker is a key contributor to calcium channel inactivation. Overall, our data are consistent with a mechanism in which inactivation of voltage-dependent calcium channels may occur via docking of the I-II linker region to a site comprising, at least in part, the domain II and III S6 segments.

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.

D1 Receptors Physically Interact with N-Type Calcium Channels to Regulate Channel Distribution and Dendritic Calcium Entry

Dopamine signaling through D1 receptors in the prefrontal cortex (PFC) plays a critical role in the maintenance of higher cognitive functions, such as working memory. At the cellular level, these functions are predicated to involve alterations in neuronal calcium levels. The dendrites of PFC neurons express D1 receptors and N-type calcium channels, yet little information exists regarding their coupling. Here, we show that D1 receptors potently inhibit N-type channels in dendrites of rat PFC neurons. Using coimmunoprecipitation, we demonstrate the existence of a D1 receptor-N-type channel signaling complex in this region, and we provide evidence for a direct receptor-channel interaction. Finally, we demonstrate the importance of this complex to receptor-channel colocalization in heterologous systems and in PFC neurons. Our data indicate that the N-type calcium channel is an important physiological target of D1 receptors and reveal a mechanism for D1 receptor-mediated regulation of cognitive function in the PFC.

Regulation of T-type calcium channels by Rho-associated kinase

We investigated the regulation of T-type channels by Rho-associated kinase (ROCK). Activation of ROCK via the endogenous ligand lysophosphatidic acid (LPA) reversibly inhibited the peak current amplitudes of rat Cav3.1 and Cav3.3 channels without affecting the voltage dependence of activation or inactivation, whereas Cav3.2 currents showed depolarizing shifts in these parameters. LPA-induced inhibition of Cav3.1 was dependent on intracellular GTP, and was antagonized by treatment with ROCK and RhoA inhibitors, LPA receptor antagonists or GDPßS. Site-directed mutagenesis of the Cav3.1 α1 subunit revealed that the ROCK-mediated effects involve two distinct phosphorylation consensus sites in the domain II-III linker. ROCK activation by LPA reduced native T-type currents in Y79 retinoblastoma and in lateral habenular neurons, and upregulated native Cav3.2 current in dorsal root ganglion neurons. Our data suggest that ROCK is an important regulator of T-type calcium channels, with potentially far-reaching implications for multiple cell functions modulated by LPA.

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.

Cross-talk between G-protein and Protein Kinase C Modulation of N-type Calcium Channels Is Dependent on the G-protein β Subunit Isoform

The modulation of N-type calcium current by protein kinases and G-proteins is a factor in the fine tuning of neurotransmitter release. We have previously shown that phosphorylation of threonine 422 in the α1B calcium channel domain I-II linker region resulted in a dramatic reduction in somatostatin receptor-mediated G-protein inhibition of the channels and that the I-II linker consequently serves as an integration center for cross-talk between protein kinase C (PKC) and G-proteins (Hamid, J., Nelson, D., Spaetgens, R., Dubel, S. J., Snutch, T. P., and Zamponi, G. W. (1999) J. Biol. Chem. 274, 6195–6202). Here we show that opioid receptor-mediated inhibition of N-type channels is affected to a lesser extent compared with that seen with somatostatin receptors, hinting at the possibility that PKC/G-protein cross-talk might be dependent on the G-protein subtype. To address this issue, we have examined the effects of four different types of G-protein β subunits on both wild type and mutant α1Bcalcium channels in which residue 422 has been replaced by glutamate to mimic PKC-dependent phosphorylation and on channels that have been directly phosphorylated by protein kinase C. Our data show that phosphorylation or mutation of residue 422 antagonizes the effect of Gβ1 on channel activity, whereas Gβ2, Gβ3, and Gβ4 are not affected. Our data therefore suggest that the observed cross-talk between G-proteins and protein kinase C modulation of N-type channels is a selective feature of the Gβ1 subunit.