Browsing by Author "Varela, Diego L."

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    D1 receptors physically interact with N-type calcium channels to regulate channel distribution and dendritic calcium entry
    (Elsevier, 2008-05-22) Kisilevsky, Alexandra E.; Mulligan, Sean J.; Altier, Christophe; Iftinca, Mircea C.; Varela, Diego L.; Tai, Chao; Chen, Lina; Hameed, Shahid; Hamid, Jawed; MacVicar, Brian Archibald; Zamponi, Gerald W.
    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.
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    Extended spectrum of idiopathic generalized epilepsies associated with CACNA1H functional variants
    (Wiley-Liss, Inc., 2007-08-14) Heron, Sarah E.; Khosravani, Houman; Varela, Diego L.; Bladen, Chris; Williams, Tristiana C.; Newman, Michelle R.; Scheffer, Ingrid; Berkovic, Samuel F.; Mulley, John C.; Zamponi, Gerald W.
    The relationship between genetic variation in the T-type calcium channel gene CACNA1H and childhood absence epilepsy is well established. The purpose of this study was to investigate the range of epilepsy syndromes for which CACNA1H variants may contribute to the genetic susceptibility architecture and determine the electrophysiological effects of these variants in relation to proposed mechanisms underlying seizures.
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    L-type calcium channel β subunit modulates angiotensin II responses in cardiomyocytes
    (Taylor and Francis, 2011-05) Hermosilla, Tamara; Moreno, Cristian; Itfinca, Mircea; Altier, Christophe; Armisén, Ricardo; Stutzin, Andrés; Zamponi, Gerald W.; Varela, Diego L.
    Angiotensin II regulation of L-type calcium currents in cardiac muscle is controversial and the underlying signaling events are not completely understood. Moreover, the possible role of auxiliary subunit composition of the channels in Angiotensin II modulation of L-type calcium channels has not yet been explored. In this work we study the role of Ca(v)β subunits and the intracellular signaling responsible for L-type calcium current modulation by Angiotensin II. In cardiomyocytes, Angiotensin II exposure induces rapid inhibition of L-type current with a magnitude that is correlated with the rate of current inactivation. Semi-quantitative PCR of cardiomyocytes at different days of culture reveals changes in the Ca(v)β subunits expression pattern that are correlated with the rate of current inactivation and with Angiotensin II effect. Over-expression of individual b subunits in heterologous systems reveals that the magnitude of Angiotensin II inhibition is dependent on the Ca(v)β subunit isoform, with Ca(v)β(1b) containing channels being more strongly regulated. Ca(v)β(2a) containing channels were insensitive to modulation and this effect was partially due to the N-terminal palmitoylation sites of this subunit. Moreover, PLC or diacylglycerol lipase inhibition prevents the Angiotensin II effect on L-type calcium channels, while PKC inhibition with chelerythrine does not, suggesting a role of arachidonic acid in this process. Finally, we show that in intact cardiomyocytes the magnitude of calcium transients on spontaneous beating cells is modulated by Angiotensin II in a Ca(v)β subunit-dependent manner. These data demonstrate that Ca(v)β subunits alter the magnitude of inhibition of L-type current by Angiotensin II.
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    Scanning mutagenesis of the I-II loop of the Cav2.2 calcium channel identifies residues Arginine 376 and Valine 416 as molecular determinants of voltage dependent G protein inhibition
    (BioMed Central Ltd., 2010-02-03) Tedford, Hugo William; Kisilevsky, Alexandra E.; Vieira, Luciene Bruno; Varela, Diego L.; Chen, Lina; Zamponi, Gerald W.
    Direct interaction with the beta subunit of the heterotrimeric G protein complex causes voltage-dependent inhibition of N-type calcium channels. To further characterize the molecular determinants of this interaction, we performed scanning mutagenesis of residues 372-387 and 410-428 of the N-type channel alpha1 subunit, in which individual residues were replaced by either alanine or cysteine. We coexpressed wild type Gbeta1gamma2 subunits with either wild type or point mutant N-type calcium channels, and voltage-dependent, G protein-mediated inhibition of the channels (VDI) was assessed using patch clamp recordings. The resulting data indicate that Arg376 and Val416 of the alpha1 subunit, residues which are surface-exposed in the presence of the calcium channel beta subunit, contribute significantly to the functional inhibition by Gbeta1. To further characterize the roles of Arg376 and Val416 in this interaction, we performed secondary mutagenesis of these residues, coexpressing the resulting mutants with wild type Gbeta1gamma2 subunits and with several isoforms of the auxiliary beta subunit of the N-type channel, again assessing VDI using patch clamp recordings. The results confirm the importance of Arg376 for G protein-mediated inhibition and show that a single amino acid substitution to phenylalanine drastically alters the abilities of auxiliary calcium channel subunits to regulate G protein inhibition of the channel.
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    Use 'em and lose 'em-activity-induced removal of calcium channels from the plasma membrane
    (Elsevier Inc., 2007-08-16) Varela, Diego L.; Zamponi, Gerald W.
    Calcium influx via L-type (Cav1.2 and Cav1.3) calcium channels is tightly regulated to ensure optimal intracellular calcium levels. Although much is known about acute modulation of these channels by second messengers, the mechanisms that control their trafficking to and from the plasma membrane remain poorly understood. In this issue of Neuron, Green and colleagues demonstrate that the opening of L-type calcium channels results in negative feedback regulation due to their calcium-dependent internalization.