Esperanza Garcia

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.

The transient receptor potential channel antagonist SKF96365 is a potent blocker of low-voltage-activated T-type calcium channels

Background and purpose:  SKF96365 (SKF), originally identified as a blocker of receptor‐mediated calcium entry, is widely used diagnostically, as a blocker of transient receptor potential canonical type (TRPC) channels. While SKF has been used as a tool to define the functional roles of TRPC channels in various cell and tissue types, there are notable overlapping physiological and pathophysiological associations between TRPC channels and low‐voltage‐activated (LVA) T‐type calcium channels. The activity of SKF against T‐type Ca channels has not been previously explored, and here we systematically investigated the effects of SKF on recombinant and native voltage‐gated Ca channel‐mediated currents.

Functional Coupling between mGluR1 and Cav3.1 T-Type Calcium Channels Contributes to Parallel Fiber-Induced Fast Calcium Signaling within Purkinje Cell Dendritic Spines

T-type voltage-gated calcium channels are expressed in the dendrites of many neurons, although their functional interactions with postsynaptic receptors and contributions to synaptic signaling are not well understood. We combine electrophysiological and ultrafast two-photon calcium imaging to demonstrate that mGluR1 activation potentiates cerebellar Purkinje cell Cav3.1 T-type currents via a G-protein- and tyrosine-phosphatase-dependent pathway. Immunohistochemical and electron microscopic investigations on wild-type and Cav3.1 gene knock-out animals show that Cav3.1 T-type channels are preferentially expressed in Purkinje cell dendritic spines and colocalize with mGluR1s. We further demonstrate that parallel fiber stimulation induces fast subthreshold calcium signaling in dendritic spines and that the synaptic Cav3.1-mediated calcium transients are potentiated by mGluR1 selectively during bursts of excitatory parallel fiber inputs. Our data identify a new fast calcium signaling pathway in Purkinje cell dendritic spines triggered by short burst of parallel fiber inputs and mediated by T-type calcium channels and mGluR1s.

Critical Residues of the Caenorhabditis elegans unc-2 Voltage-Gated Calcium Channel That Affect Behavioral and Physiological Properties

The Caenorhabditis elegans unc-2 gene encodes a voltage-gated calcium channel α1 subunit structurally related to mammalian dihydropyridine-insensitive high-threshold channels. In the present paper we describe the characterization of seven alleles of unc-2. Using an unc-2 promoter-tagged green fluorescent protein construct, we show that unc-2 is primarily expressed in motor neurons, several subsets of sensory neurons, and the HSN and VC neurons that control egg laying. Examination of behavioral phenotypes, including defecation, thrashing, and sensitivities to aldicarb and nicotine suggests that UNC-2 acts presynaptically to mediate both cholinergic and GABAergic neurotransmission. Sequence analysis of the unc-2 alleles shows that e55, ra605, ra606, ra609, and ra610 all are predicted to prematurely terminate and greatly reduce or eliminate unc-2 function. In contrast, the ra612 and ra614 alleles are missense mutations resulting in the substitution of highly conserved residues in the C terminus and the domain IVS4-IVS5 linker, respectively. Heterologous expression of a rat brain P/Q-type channel containing the ra612 mutation shows that the glycine to arginine substitution affects a variety of channel characteristics, including the voltage dependence of activation, steady-state inactivation, as well as channel kinetics. Overall, our findings suggest that UNC-2 plays a pivotal role in mediating a number of physiological processes in the nematode and also defines a number of critical residues important for calcium channel function in vivo.

Contribution of calcium-dependent facilitation to synaptic plasticity revealed by migraine mutations in the P/Q-type calcium channel

The dynamics, computational power, and strength of neural circuits are essential for encoding and processing information in the CNS and rely on short and long forms of synaptic plasticity. In a model system, residual calcium (Ca2+) in presynaptic terminals can act through neuronal Ca2+ sensor proteins to cause Ca2+-dependent facilitation (CDF) of P/Q-type channels and induce short-term synaptic facilitation. However, whether this is a general mechanism of plasticity at intact central synapses and whether mutations associated with human disease affect this process have not been described to our knowledge. In this report, we find that, in both exogenous and native preparations, gain-of-function missense mutations underlying Familial Hemiplegic Migraine type 1 (FHM-1) occlude CDF of P/Q-type Ca2+ channels. In FHM-1 mutant mice, the alteration of P/Q-type channel CDF correlates with reduced short-term synaptic facilitation at cerebellar parallel fiber-to-Purkinje cell synapses. Two-photon imaging suggests that P/Q-type channels at parallel fiber terminals in FHM-1 mice are in a basally facilitated state. Overall, the results provide evidence that FHM-1 mutations directly affect both P/Q-type channel CDF and synaptic plasticity and that together likely contribute toward the pathophysiology underlying FHM-1. The findings also suggest that P/Q-type channel CDF is an important mechanism required for normal synaptic plasticity at a fast synapse in the mammalian CNS.

Ca(V)2.1 P/Q-type calcium channel alternative splicing affects the functional impact of familial hemiplegic migraine mutations: implications for calcium channelopathies.

Alternative splicing is known to generate multiple functionally distinct calcium channel variants that exhibit distinct spatial and temporal expression patterns. In humans, naturally occurring mutations in genes encoding calcium channel pore forming α1-subunits are associated with several severe hereditary disorders although it remains to be described whether there exists any relationship between the physiological effects of these mutations and calcium channel splice variation. In the present study, we systematically compare the biophysical effects of three type-1 familial hemiplegic migraine (FHM-1) mutations in two predominant splice variants of the neuronal CaV2.1 P/Q-type channel. All three FHM-1 mutations cause a greater hyperpolarizing shift in voltage-dependent properties when expressed in the short carboxyl terminus variant (CaV2.1 Δ47) compared to the long variant (CaV2.1 +47). Furthermore, the FHM-1 mutations also exhibit differential splice variant-specific effects on recovery from inactivation and accumulation of inactivation during tonic and burst firing. Our findings provide important insight concerning the role of calcium channel alternatively spliced variants and the molecular pathophysiology of FHM-1 and potentially of other calcium channelopathies.

Selective Inhibition of Cav3.3 T-type Calcium Channels by Gαq/11-coupled Muscarinic Acetylcholine Receptors

T-type calcium channels play critical roles in controlling neuronal excitability, including the generation of complex spiking patterns and the modulation of synaptic plasticity, although the mechanisms and extent to which T-type Ca2+ channels are modulated by G-protein-coupled receptors (GPCRs) remain largely unexplored. To examine specific interactions between T-type Ca2+ channel subtypes and muscarinic acetylcholine receptors (mAChRS), the Cav3.1 (α1G), Cav3.2 (α1H), and Cav3.3 (α) T-type Ca2+1Ichannels were co-expressed with the M1 Gαq/11-coupled mAChR. Perforated patch recordings demonstrate that activation of M1 receptors has a strong inhibitory effect on Cav3.3 T-type Ca2+ currents but either no effect or a moderate stimulating effect on Cav3.1 and Cav3.2 peak current amplitudes. This differential modulation was observed for both rat and human T-type Ca2+ channel variants. The inhibition of Cav3.3 channels by M1 receptors is reversible, use-independent, and associated with a concomitant increase in inactivation kinetics. Loss-of-function experiments with genetically encoded antagonists of Gα and Gβγ proteins and gain-of-function experiments with genetically encoded Gα subtypes indicate that M1 receptor-mediated inhibition of Cav3.3 occurs through Gαq/11. This is supported by experiments showing that activation of the M3 and M5 Gαq/11-coupled mAChRs also causes inhibition of Cav3.3 currents, although Gαi-coupled mAChRs (M2 and M4) have no effect. Examining Cav3.1-Cav3.3 chimeric channels demonstrates that two distinct regions of the Cav3.3 channel are necessary and sufficient for complete M1 receptor-mediated channel inhibition and represent novel sites not previously implicated in T-type channel modulation.

Splice-variant changes of the Ca(V)3.2 T-type calcium channel mediate voltage-dependent facilitation and associate with cardiac hypertrophy and development.

Low voltage-activated T-type calcium (Ca) channels contribute to the normal development of the heart and are also implicated in pathophysiological states such as cardiac hypertrophy. Functionally distinct T-type Ca channel isoforms can be generated by alternative splicing from each of three different T-type genes (CaV3.1, CaV3.2,CaV3 .3), although it remains to be described whether specific splice variants are associated with developmental states and pathological conditions. We aimed to identify and functionally characterize CaV3.2 T-type Ca channel alternatively spliced variants from newborn animals and to compare with adult normotensive and spontaneously hypertensive rats (SHR). DNA sequence analysis of full-length CaV3.2 cDNA generated from newborn heart tissue identified ten major regions of alternative splicing, the more common variants of which were analyzed by quantitative real-time PCR (qRT-PCR) and also subject to functional examination by whole-cell patch clamp. The main findings are that: (1) cardiac CaV3.2 T-type Ca channels are subject to considerable alternative splicing, (2) there is preferential expression ofCaV3 .2(-25) splice variant channels in newborn rat heart with a developmental shift in adult heart that results in approximately equal levels of expression of both (+25) and (-25) exon variants, (3) in the adult stage of hypertensive rats there is a both an increase in overallCaV3 .2 expression and a shift towards expression of CaV3.2(+25) containing channels as the predominant form, and (4) alternative splicing confers a variant-specific voltage-dependent facilitation ofCaV3 .2 channels. We conclude that CaV3.2 alternative splicing generates significant T-type Ca channel structural and functional diversity with potential implications relevant to cardiac developmental and pathophysiological states.

In vivo imaging reveals that pregabalin inhibits cortical spreading depression and propagation to subcortical brain structures

Significance Spreading depression is proposed to underlie migraine with aura, a type of debilitating headache for which few pharmacological treatments are available. The pain drug pregabalin has demonstrated initial promising results for the treatment of migraine in the clinic. Utilizing animal models of congenital migraine and live brain imaging, we describe the cortical and subcortical migration of the spreading depression wave. Further, pregabalin is shown to be effective at suppressing spreading depression initiation, wave speed, and subcortical propagation, and also to affect nerve cell signalling directly. Overall, the study supports the therapeutic potential of pregabalin in both noncongenital migraineurs and patients with mild congenital migraine. Migraine is characterized by severe headaches that can be preceded by an aura likely caused by cortical spreading depression (SD). The antiepileptic pregabalin (Lyrica) shows clinical promise for migraine therapy, although its efficacy and mechanism of action are unclear. As detected by diffusion-weighted MRI (DW-MRI) in wild-type (WT) mice, the acute systemic administration of pregabalin increased the threshold for SD initiation in vivo. In familial hemiplegic migraine type 1 mutant mice expressing human mutations (R192Q and S218L) in the CaV2.1 (P/Q-type) calcium channel subunit, pregabalin slowed the speed of SD propagation in vivo. Acute systemic administration of pregabalin in vivo also selectively prevented the migration of SD into subcortical striatal and hippocampal regions in the R192Q strain that exhibits a milder phenotype and gain of CaV2.1 channel function. At the cellular level, pregabalin inhibited glutamatergic synaptic transmission differentially in WT, R192Q, and S218L mice. The study describes a DW-MRI analysis method for tracking the progression of SD and provides support and a mechanism of action for pregabalin as a possible effective therapy in the treatment of migraine.

Peripheral nerve injury increases contribution of L-type calcium channels to synaptic transmission in spinal lamina II: Role of α2δ–1 subunits:

Background Following peripheral nerve chronic constriction injury, the accumulation of the α2δ–1 auxiliary subunit of voltage-gated Ca2+ channels in primary afferent terminals contributes to the onset of neuropathic pain. Overexpression of α2δ–1 in Xenopus oocytes increases the opening properties of Cav1.2 L-type channels and allows Ca2+ influx at physiological membrane potentials. We therefore posited that L-type channels play a role in neurotransmitter release in the superficial dorsal horn in the chronic constriction injury model of neuropathic pain. Results Whole-cell recording from lamina II neurons from rats, subject to sciatic chronic constriction injury, showed that the L-type Ca2+ channel blocker, nitrendipine (2 µM) reduced the frequency of spontaneous excitatory postsynaptic currents. Nitrendipine had little or no effect on spontaneous excitatory postsynaptic current frequency in neurons from sham-operated animals. To determine whether α2δ–1 is involved in upregulating function of Cav1.2 L-type channels, we tested the effect of the α2δ–1 ligand, gabapentin (100 µM) on currents recorded from HEK293F cells expressing Cav1.2/β4/α2δ–1 channels and found a significant decrease in peak amplitude with no effect on control Cav1.2/β4/α2δ–3 expressing cells. In PC-12 cells, gabapentin also significantly reduced the endogenous dihydropyridine-sensitive calcium current. In lamina II, gabapentin reduced spontaneous excitatory postsynaptic current frequency in neurons from animals subject to chronic constriction injury but not in those from sham-operated animals. Intraperitoneal injection of 5 mg/kg nitrendipine increased paw withdrawal threshold in animals subject to chronic constriction injury. Conclusion We suggest that L-type channels show an increased contribution to synaptic transmission in lamina II dorsal horn following peripheral nerve injury. The effect of gabapentin on Cav1.2 via α2δ–1 may contribute to its anti-allodynic action.