Ivana A. Souza

Role of Prelimbic GABAergic Circuits in Sensory and Emotional Aspects of Neuropathic Pain

Noxious stimuli are detected by peripheral nociceptors and then transmitted to higher CNS centers, where they are perceived as an unpleasant sensation. The mechanisms that govern the emotional component associated with pain are still incompletely understood. Here, we used optogenetic approaches both in vitro and in vivo to address this issue. We found that peripheral nerve injury inhibits pyramidal cell firing in the prelimbic area of the prefrontal cortex as a result of feed-forward inhibition mediated by parvalbumin-expressing GABAergic interneurons. In addition, activation of inhibitory archaerhodopsin or excitatory channelrhodopsin-2 in these neurons decreased and increased pain responses, respectively, in freely moving mice and accordingly modulated conditioned place preference scores and place escape/avoidance behavior. Our findings thus demonstrate an important role of the prelimbic area in sensory and emotional aspects of pain and identify GABAergic circuits in this region as a potential target for pain therapeutics.

Block of T-type calcium channels by protoxins I and II

BackgroundLow-voltage-activated (T-type) calcium channels play a crucial role in a number of physiological processes, including neuronal and cardiac pacemaker activity and nociception. Therefore, finding specific modulators and/or blockers of T-type channels has become an important field of drug discovery. One characteristic of T-type calcium channels is that they share several structural similarities with voltage-gated sodium channels (VGSCs). We therefore hypothesized that binding sites for certain sodium channel blocking peptide toxins may be present in T-type calcium channels.FindingsThe sodium channel blocker ProTx I tonically blocked native and transiently expressed T-type channels in the sub- to low micro molar range with at least a ten-fold selectivity for the T-type calcium channel hCav3.1 over hCav3.3, and more than one hundred fold selectivity over hCav3.2. Using chimeras of hCav3.1 and hCav3.3, we determined that the domain IV region of hCav3.1 is a major determinant of toxin affinity, with a minor contribution from domain II. Further analysis revealed several residues in a highly conserved region between T-type and sodium channels that may correspond to toxin binding sites. Mutagenesis of several of these residues on an individual basis, however, did not alter the blocking effects of the toxin. ProTx II on the other hand preferentially blocked hCav3.2 and significantly shifted the steady state inactivation of this channel.ConclusionsProTx I blocks hCav3.1 both selectively and with high affinity. Domain IV appears to play a major role in this selectivity with some contribution from domain II. Given the structural similarities between sodium and T-type calcium channels and the apparent conservation in toxin binding sites, these data could provide insights into the development and synthesis of novel T-type channel antagonists.

A novel calmodulin site in the Cav1.2 N-terminus regulates calcium-dependent inactivation

The L-type voltage-gated calcium channel Cav1.2 is important for excitation-contraction coupling in the heart, as well as CREB-mediated transcription in the brain. The ubiquitous calcium-binding protein calmodulin (CaM) is known to modulate calcium-dependent inactivation (CDI) of these channels, thus limiting the amount of calcium entering via Cav1.2 during prolonged or repetitive membrane depolarizations. The proximal N-terminus of Cav1.2 contains a CaM-binding site at residue W52 that is critical for a type of CDI that is mediated by the N-terminal lobe of CaM. Here, we identify a second CaM interaction site in the Cav1.2 N-terminus downstream of the W52 site that is formed by residue C106. We show by site-directed mutagenesis coupled with electrophysiological measurements that this region of the channel functionally partakes in N-lobe CDI, likely by acting as a gating transduction motif. Thus, our data indicate that calcium regulation of Cav1.2 channels is more complex than previously thought, and involves more than one region within the channels N-terminal domain.

The Cav1.2 N terminus contains a CaM kinase site that modulates channel trafficking and function

The L-type voltage-gated calcium channel Cav1.2 and the calcium-activated CaM kinase cascade both regulate excitation transcription coupling in the brain. CaM kinase is known to associate with the C terminus of Cav1.2 in a region called the PreIQ-IQ domain, which also binds multiple calmodulin molecules. Here we identify and characterize a second CaMKII binding site in the N terminus of Cav1.2 that is formed by a stretch of four amino residues (cysteine–isoleucine–serine–isoleucine) and which regulates channel expression and function. By using live cell imaging of tsA-201 cells we show that GFP fusion constructs of the CaMKII binding region, termed N2B-II co-localize with mCherry-CaMKII. Mutating CISI to AAAA ablates binding to and colocalization with CaMKII. Cav1.2-AAAA channels show reduced cell surface expression in tsA-201 cells, but interestingly, display an increase in channel function that offsets the trafficking deficit. Altogether our data reveal that the proximal N terminus of Cav1.2 contains a CaMKII binding region which contributes to channel surface expression and function.

Effect of the Brugada syndrome mutation A39V on calmodulin regulation of Cav1.2 channels

BackgroundThe L-type calcium channel Cav1.2 is important for brain and heart function. The ubiquitous calcium sensing protein calmodulin (CaM) regulates calcium dependent gating of Cav1.2 channels by reducing calcium influx, a process known as calcium-dependent inactivation (CDI). Dissecting the calcium-dependence of CaM in this process has benefited greatly from the use of mutant CaM molecules which are unable to bind calcium to their low affinity (N-lobe) and high affinity (C-lobe) binding sites. Unlike CDI, it is unknown whether CaM can modulate the activation gating of Cav1.2 channels.ResultsWe examined a Cav1.2 point mutant in the N-terminus region of the channel (A39V) that has been previously linked to Brugada syndrome. Using mutant CaM constructs in which the N- and/or C-lobe calcium binding sites were ablated, we were able to show that this Brugada syndrome mutation disrupts N-lobe CDI of the channel. In the course of these experiments, we discovered that all mutant CaM molecules were able to alter the kinetics of channel activation even in the absence of calcium for WT-Cav1.2, but not A39V-Cav1.2 channels. Moreover, CaM mutants differentially shifted the voltage-dependence of activation for WT and A39V-Cav1.2 channels to hyperpolarized potentials. Our data therefore suggest that structural changes in CaM that arise directly from site directed mutagenesis of calcium binding domains alter activation gating of Cav1.2 channels independently of their effects on calcium binding, and that the N-terminus of the channel contributes to this CaM dependent process.ConclusionsOur data indicate that caution must be exercised when interpreting the effects of CaM mutants on ion channel gating.

Identification of interleukin-1 beta as a key mediator in the upregulation of Cav3.2–USP5 interactions in the pain pathway

We recently reported that nerve injury or peripheral inflammation triggers an upregulation of the deubiquitinase, USP5 in mouse dorsal root ganglion and spinal dorsal horn. This leads to dysregulated ubiquitination of Cav3.2 T-type calcium channels, thus increasing Cav3.2 channel plasma membrane expression and nociceptive signaling in the primary afferent pain pathway. This phenomenon could be recapitulated by noninvasive, optogenetic activation of transient receptor potential vanilloid-1–expressing nociceptors, indicating that neuronal activity is a key player in this process. Given the relevance of the pro-inflammatory cytokine interleukin-1 beta in many forms of pathological pain, we hypothesized that interleukin-1 beta may be a critical cofactor required to drive upregulation of interactions between USP5 and Cav3.2 channels. Here, we report that gene expression, as well as protein levels for interleukin-1 beta and the endogenous interleukin-1 receptor-I antagonist, IL-1Ra are unaltered following conditioning stimulation of optogenetically targeted cutaneous nociceptors, indicating that neuronal activity is not a driver of interleukin-1 beta signaling. In contrast, co-immunoprecipitation experiments revealed that intrathecal administration of interleukin-1 beta in wild-type mice led to an increase in the interaction between USP5 and Cav3.2 in the spinal dorsal horn. Moreover, disruption of the interaction between USP5 and Cav3.2 with TAT peptides suppressed acute nocifensive responses produced by interleukin-1 beta, which was similar to that achieved by elimination of T-type channel activity with the channel blockers, mibefradil, or TTA-A2. Finally, this upregulation could be maintained in dorsal root ganglion neuron cultures exposed overnight to interleukin-1 beta, while the copresence of interleukin-1 receptor antagonist or the dampening of neuronal cell activity with tetrodotoxin attenuated this response. Altogether, our findings identify interleukin-1 beta as an upstream trigger for the upregulation of interactions between USP5 and Cav3.2 channels in the pain pathway, presumably by triggering increased firing activity in afferent fibers.

Reduced Hyperpolarization-Activated Current Contributes to Enhanced Intrinsic Excitability in Cultured Hippocampal Neurons from PrP(-/-) Mice.

Genetic ablation of cellular prion protein (PrPC) has been linked to increased neuronal excitability and synaptic activity in the hippocampus. We have previously shown that synaptic activity in hippocampi of PrP-null mice is increased due to enhanced N-methyl-D-aspartate receptor (NMDAR) function. Here, we focused on the effect of PRNP gene knock-out (KO) on intrinsic neuronal excitability, and in particular, the underlying ionic mechanism in hippocampal neurons cultured from P0 mouse pups. We found that the absence of PrPC profoundly affected the firing properties of cultured hippocampal neurons in the presence of synaptic blockers. The membrane impedance was greater in PrP-null neurons, and this difference was abolished by the hyperpolarization-activated cyclic nucleotide-gated (HCN) channel blocker ZD7288 (100 μM). HCN channel activity appeared to be functionally regulated by PrPC. The amplitude of voltage sag, a characteristic of activating HCN channel current (Ih), was decreased in null mice. Moreover, Ih peak current was reduced, along with a hyperpolarizing shift in activation gating and slower kinetics. However, neither HCN1 nor HCN2 formed a biochemical complex with PrPC. These results suggest that the absence of PrP downregulates the activity of HCN channels through activation of a cell signaling pathway rather than through direct interactions. This in turn contributes to an increase in membrane impedance to potentiate neuronal excitability.

The amino-terminus of high voltage activated calcium channels: CaM you or can't you?

Voltage-gated calcium channels (VGCCs), calmodulin (CaM), and calmodulin kinase II (CaMKII) are essential for various nervous system functions. CaM and CaMKII differentially regulate calcium dependent facilitation (CDF) and calcium dependent inactivation (CDI) of the Cav1 and Cav2 families of VGCCs. It is generally accepted that conserved structures in the C-terminus of these channels regulate CDF and CDI, and yet recent evidence indicates that other intracellular regions may be involved. We recently discovered that N-terminal sequences in Cav1.2 bind CaM and CaMKII, and function to regulate CDI as well as surface expression and open probability, respectively. Cav1 and Cav2 share significant portions of N-terminal sequence and therefore we explored whether homologous binding sites might exist in Cav2.1. Here, we show that like the proximal N-terminus of Cav1.2, the homologous region of Cav2.1 contains sequences which interact either directly or indirectly with CaM.

Cav3.1 overexpression is associated with negative characteristics and prognosis in non-small cell lung cancer

Introduction Voltage-gated calcium channels (VGCC) have been found to be differentially expressed in several different tumor types, but their role in tumor growth, malignant invasion, metastases and impact on clinical outcomes has not been clarified. Materials and Methods From a cohort database of 193 patients with early-stage NSCLC, 163 formalin-fixed paraffin-embedded specimens were available for analysis to construct tissue microarrays. Cav3.1 protein expression was detected using fluorescence immunohistochemistry, and quantified using automated image acquisition and analysis. Results Among the cohort of 193 NSCLC patients, adenocarcinoma (53.9%) and squamous cell carcinoma (SCC) (30.1%) were the most common histologies. There was no difference between SCC and non-SCC subtypes in overall survival (OS) or relapse-free survival (RFS); 74.2 vs 90.1 months (p = 0.543) and 48.8 vs 52.6 months (p = 0.766), respectively. T-type VGCC 3.1 (Cav3.1) overexpression was assessed by tissue microarray immunohistochemistry analysis from 163 available patient samples. Eighteen (11.0%) NSCLC primaries were found to have Cav3.1 overexpression levels, and were significantly associated with SCC histology (p < 0.001), larger tumor size (p < 0.001) and later stage disease at diagnosis (p = 0.019). Median OS was 48.6 vs 106.7 months for Cav3.1 overexpressing and non-overexpressing patients, respectively (p = 0.032). Regression analysis revealed a significantly negative effect for Cav3.1 overexpression on RFS (Hazard ratio [HR] = 2.02, p = 0.048). Conclusions Cav3.1 overexpression is a potential biomarker for poorer patient outcomes. These results bring supportive evidence for calcium channels inducing an aggressive phenotype in NSCLC and potentially may serve as a therapeutic target in overexpressing tumors.

Down-regulation of T-type Cav3.2 channels by hyperpolarization-activated cyclic nucleotide-gated channel 1 (HCN1): Evidence of a signaling complex

ABSTRACT Formation of complexes between ion channels is important for signal processing in the brain. Here we investigate the biochemical and biophysical interactions between HCN1 channels and Cav3.2 T-type channels. We found that HCN1 co-immunoprecipitated with Cav3.2 from lysates of either mouse brain or tsA-201 cells, with the HCN1 N-terminus associating with the Cav3.2 N-terminus. Cav3.2 channel activity appeared to be functionally regulated by HCN1. The expression of HCN1 induced a decrease in Cav3.2 Ba2+ influx (IBa2+) along with altered channel kinetics and a depolarizing shift in activation gating. However, a reciprocal regulation of HCN1 by Cav3.2 was not observed. This study highlights a regulatory role of HCN1 on Cav3.2 voltage-dependent properties, which are expected to affect physiologic functions such as synaptic transmission and cellular excitability.