Alternative splicing of the rat Cav3.3 T-type calcium channel gene produces variants with distinct functional properties11The sequences reported herein have been assigned GenBank accession numbers AY128644–AY128648.

AbstractMolecular diversity in T-type Ca2+ channels is produced by expression of three genes, and alternative splicing of those genes. Prompted by differences noted between rat and human Cav3.3 sequences, we searched for splice variants. We cloned six variants, which are produced by splicing at exon 33 and exon 34. Expression of the variants differed between brain regions. The electrophysiological properties of the variants displayed similar voltage-dependent gating, but differed in their kinetic properties. The functional impact of splicing was inter-related, suggesting an interaction. We conclude that alternative splicing of the Cav3.3 gene produces channels with distinct properties

I–II Loop Structural Determinants in the Gating and Surface Expression of Low Voltage-Activated Calcium Channels

The intracellular loops that interlink the four transmembrane domains of Ca2+- and Na+-channels (Cav, Nav) have critical roles in numerous forms of channel regulation. In particular, the intracellular loop that joins repeats I and II (I–II loop) in high voltage-activated (HVA) Ca2+ channels possesses the binding site for Cavβ subunits and plays significant roles in channel function, including trafficking the α1 subunits of HVA channels to the plasma membrane and channel gating. Although there is considerable divergence in the primary sequence of the I–II loop of Cav1/Cav2 HVA channels and Cav3 LVA/T-type channels, evidence for a regulatory role of the I–II loop in T-channel function has recently emerged for Cav3.2 channels. In order to provide a comprehensive view of the role this intracellular region may play in the gating and surface expression in Cav3 channels, we have performed a structure-function analysis of the I–II loop in Cav3.1 and Cav3.3 channels using selective deletion mutants. Here we show the first 60 amino acids of the loop (post IS6) are involved in Cav3.1 and Cav3.3 channel gating and kinetics, which establishes a conserved property of this locus for all Cav3 channels. In contrast to findings in Cav3.2, deletion of the central region of the I–II loop in Cav3.1 and Cav3.3 yielded a modest increase (+30%) and a reduction (−30%) in current density and surface expression, respectively. These experiments enrich our understanding of the structural determinants involved in Cav3 function by highlighting the unique role played by the intracellular I–II loop in Cav3.2 channel trafficking, and illustrating the prominent role of the gating brake in setting the slow and distinctive slow activation kinetics of Cav3.3

Orientation of the Calcium Channel β Relative to the α12.2 Subunit Is Critical for Its Regulation of Channel Activity

BACKGROUND: The Ca(v)beta subunits of high voltage-activated Ca(2+) channels control the trafficking and biophysical properties of the alpha(1) subunit. The Ca(v)beta-alpha(1) interaction site has been mapped by crystallographic studies. Nevertheless, how this interaction leads to channel regulation has not been determined. One hypothesis is that betas regulate channel gating by modulating movements of IS6. A key requirement for this direct-coupling model is that the linker connecting IS6 to the alpha-interaction domain (AID) be a rigid structure. METHODOLOGY/PRINCIPAL FINDINGS: The present study tests this hypothesis by altering the flexibility and orientation of this region in alpha(1)2.2, then testing for Ca(v)beta regulation using whole cell patch clamp electrophysiology. Flexibility was induced by replacement of the middle six amino acids of the IS6-AID linker with glycine (PG6). This mutation abolished beta2a and beta3 subunits ability to shift the voltage dependence of activation and inactivation, and the ability of beta2a to produce non-inactivating currents. Orientation of Ca(v)beta with respect to alpha(1)2.2 was altered by deletion of 1, 2, or 3 amino acids from the IS6-AID linker (Bdel1, Bdel2, Bdel3, respectively). Again, the ability of Ca(v)beta subunits to regulate these biophysical properties were totally abolished in the Bdel1 and Bdel3 mutants. Functional regulation by Ca(v)beta subunits was rescued in the Bdel2 mutant, indicating that this part of the linker forms beta-sheet. The orientation of beta with respect to alpha was confirmed by the bimolecular fluorescence complementation assay. CONCLUSIONS/SIGNIFICANCE: These results show that the orientation of the Ca(v)beta subunit relative to the alpha(1)2.2 subunit is critical, and suggests additional points of contact between these subunits are required for Ca(v)beta to regulate channel activity

El glutamato en la retina de los vertebrados: de los receptores membranales a la regulación génica

In this brief review, the function of the excitatory amino acids glutamate and aspartate as neurotransmitters in the nervous system and the vertebrate retina is analyzed. These compounds induce a whole range of cellular responses, due to the presence of distinct types of receptors in postsynaptic cells, showing differences in structure as well as signal transduction mechanisms such as ligand-gating of ion channels or activation of second messenger cascades. The characteristics of these receptors are described in the light of molecular biology studies, and their distribution in neurons and glia from the retina is also documented. Attention is drawn to the recent finding of glial cell excitability in response to neurotransmitters, which opens a new line of cell communication within the nervous system

Cloning and expression of the human T-type channel Ca(v)3.3: insights into prepulse facilitation.

The full-length human Ca(v)3.3 (alpha(1I)) T-type channel was cloned, and found to be longer than previously reported. Comparison of the cDNA sequence to the human genomic sequence indicates the presence of an additional 4-kb exon that adds 214 amino acids to the carboxyl terminus and encodes the 3' untranslated region. The electrophysiological properties of the full-length channel were studied after transient transfection into 293 human embryonic kidney cells using 5 mM Ca(2+) as charge carrier. From a holding potential of -100 mV, step depolarizations elicited inward currents with an apparent threshold of -70 mV, a peak of -30 mV, and reversed at +40 mV. The kinetics of channel activation, inactivation, deactivation, and recovery from inactivation were very similar to those reported previously for rat Ca(v)3.3. Similar voltage-dependent gating and kinetics were found for truncated versions of human Ca(v)3.3, which lack either 118 or 288 of the 490 amino acids that compose the carboxyl terminus. A major difference between these constructs was that the full-length isoform generated twofold more current. These results suggest that sequences in the distal portion of Ca(v)3.3 play a role in channel expression. Studies on the voltage-dependence of activation revealed that a fraction of channels did not gate as low voltage-activated channels, requiring stronger depolarizations to open. A strong depolarizing prepulse (+100 mV, 200 ms) increased the fraction of channels that gated at low voltages. In contrast, human Ca(v)3.3 isoforms with shorter carboxyl termini were less affected by a prepulse. Therefore, Ca(v)3.3 is similar to high voltage-activated Ca(2+) channels in that depolarizing prepulses can regulate their activity, and their carboxy termini play a role in modulating channel activity

Peripheral and spinal TRPA1 channels contribute to formalin-induced long-lasting mechanical hypersensitivity

Vladimir A Martínez-Rojas,1 Guadalupe García,1 Roxana Noriega-Navarro,1 Crystell G Guzmán-Priego,2 Jorge E Torres-López,2,3 Vinicio Granados-Soto,4 Janet Murbartián1 1Departamento de Farmacobiología, Centro de Investigación y de Estudios Avanzados (Cinvestav), Unidad Coapa, Ciudad de México, 2Laboratorio Mecanismos del Dolor, Centro de Investigación, División Académica de Ciencias de la Salud, Universidad Juárez Autónoma de Tabasco, 3Hospital Regional de Alta Especialidad “Dr. Juan Graham Casasús”, Villahermosa, Tabasco, 4Neurobiology of Pain Laboratory, Departamento de Farmacobiología, Cinvestav, Unidad Coapa, Ciudad de México, México Background: Transient receptor potential ankyrin 1 (TRPA1) is a non-selective cation channel expressed by a subset of nociceptive neurons that acts as a multimodal receptor. Its activity contributes to modulate nociceptive transmission in acute inflammatory pain. However, the role of this channel in chronic pain has been less studied. The purpose of this study was to investigate the local peripheral and spinal participation of TRPA1 channels in formalin-induced long-lasting hypersensitivity.Materials and methods: Formalin (1%)-induced chronic hypersensitivity was determined by the application of von Frey filaments to ipsilateral and contralateral paws and through pharmacological testing using a selective TRPA1 blocker (A-967079). TRPA1 expression in the dorsal root ganglion (DRG) and spinal cord was analyzed by Western blotting.Results: Formalin (1%) injection produced acute flinching behavior (1 h) as well as secondary allodynia and hyperalgesia (12 days). Local peripheral pretreatment (10 min before) or posttreatment (6 days later) with A-967079 (1–100 µM) partially prevented and reversed, respectively, in a dose-dependent manner, long-lasting secondary mechanical allodynia and hyperalgesia evoked by 1% formalin. Likewise, intrathecal pretreatment or posttreatment with A-967079 partially prevented and reversed, respectively, formalin-induced long-lasting hypersensitivity. A-967079 (100 µM) completely abolished the pro-nociceptive effect of formalin (adjusted to pH 7.4). Finally, formalin injection increased TRPA1 protein expression in the DRG and spinal cord.Conclusion: Results indicate that TRPA1 expressed in the DRG and spinal cord plays a relevant role in formalin-induced long-lasting secondary nociceptive hypersensitivity. Keywords: allodynia, chronic pain, formalin, hyperalgesia, TRPA

Electrophysiological properties of WT, PG6, and PA6 channels and their regulation by β2a and β3.

<p>The values of V₅₀ and k were calculated for each cell, then averaged. R values determined from test pulses to +20 mV. Data shown are mean±SEM from the number of cells shown in parentheses. Statistical significance of the β2a and β3 effects relative to either α₁ alone (+α₂δ1) were determined using ANOVA.</p>†<p>Currents from PG6 were completely inactivated by 350 ms, so the residual current at 25 ms (divided by peak) is reported (R₂₅).</p>§<p>Current density was estimated from the peak of the <i>I–V</i> curve, and statistical significance was determined using Student's <i>t</i>-test.</p><p>*P<0.05, **P<0.01.</p

Introduction of the poly-glycine substitution in the α₁2.2 subunit disrupts (PG6), while poly-alanine substitution (PA6) preserves Ca_vβ regulation.

<p>Panels A–D show data obtained with PG6, while panels E–H show data obtained with PA6. (A, E) Peak current-voltage relationships normalized to cell capacitance for the respective α₁ mutant expressed alone or with β2a or β3. (B, F) Activation represented by the normalized conductance (G/G_max). The residual current after either 25 ms (C) or 350 ms (G) of depolarization divided by the maximum inward current, and plotted against test potential. Representative traces normalized to the peak inward current are shown in the inset. (D, H) Comparison of the β2a and β3 effects on steady-state inactivation estimated using 15 s prepulses to varying potentials. Dotted lines represent steady-state inactivation measured for WT channels in the presence of β3 (α₁2.2+α₂δ1+β3).</p