RESUMO
T-type calcium channels are essential contributors to the transmission of nociceptive signals in the primary afferent pain pathway. Here, we show that T-type calcium channels are ubiquitinated by WWP1, a plasma-membrane-associated ubiquitin ligase that binds to the intracellular domain III-IV linker region of the Cav3.2 T-type channel and modifies specific lysine residues in this region. A proteomic screen identified the deubiquitinating enzyme USP5 as a Cav3.2 III-IV linker interacting partner. Knockdown of USP5 via shRNA increases Cav3.2 ubiquitination, decreases Cav3.2 protein levels, and reduces Cav3.2 whole-cell currents. In vivo knockdown of USP5 or uncoupling USP5 from native Cav3.2 channels via intrathecal delivery of Tat peptides mediates analgesia in both inflammatory and neuropathic mouse models of mechanical hypersensitivity. Altogether, our experiments reveal a cell signaling pathway that regulates T-type channel activity and their role in nociceptive signaling.
Assuntos
Canais de Cálcio Tipo T/metabolismo , Endopeptidases/metabolismo , Inflamação/fisiopatologia , Neuralgia/enzimologia , Animais , Canais de Cálcio Tipo T/genética , Células Cultivadas , Modelos Animais de Doenças , Endopeptidases/genética , Adjuvante de Freund/toxicidade , Humanos , Hiperalgesia/diagnóstico , Hiperalgesia/fisiopatologia , Técnicas In Vitro , Inflamação/induzido quimicamente , Masculino , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/genética , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteínas do Tecido Nervoso/metabolismo , Neuralgia/tratamento farmacológico , Limiar da Dor/efeitos dos fármacos , Limiar da Dor/fisiologia , Peptídeos/uso terapêutico , Células Receptoras Sensoriais/efeitos dos fármacos , Células Receptoras Sensoriais/fisiologia , Medula Espinal/citologia , Transfecção , Ubiquitinação/genética , Ubiquitinação/fisiologiaRESUMO
When transiently expressed in tsA-201 cells, Ca(v)1.4 calcium channels support only modest whole-cell currents with unusually slow voltage-dependent inactivation kinetics. To examine the basis for this unique behavior we used cell-attached patch single-channel recordings using 100 mM external barium as the charge carrier to determine the single-channel properties of Ca(v)1.4 and to compare them to those of the Ca(v)1.2. Ca(v)1.4 channel openings occurred infrequently and were of brief duration. Moreover, openings occurred throughout the duration of the test depolarization, indicating that the slow inactivation kinetics observed at the whole-cell level are caused by sustained channel activity. Ca(v)1.4 and Ca(v)1.2 channels displayed similar latencies to first opening. Because of the rare occurrence of events, the probability of opening could not be precisely determined but was estimated to be <0.015 over a voltage range of -20 to +20 mV. The single-channel conductance of Ca(v)1.4 channels was approximately 4 pS compared with approximately 20 pS for Ca(v)1.2 under the same experimental conditions. Additionally, in the absence of divalent cations, Ca(v)1.4 channels pass cesium ions with a single-channel conductance of approximately 21 pS. Although Ca(v)1.2 opening events were best described kinetically with two open time constants, Ca(v)1.4 open times were best described by a single time constant. BayK8644 slightly enhanced the single-channel conductance in addition to increasing the open time constant for Ca(v)1.4 channels by approximately 45% without, however, causing the appearance of an additional slower gating mode. Overall, our data indicate that single Ca(v)1.4 channels support only minute amounts of calcium entry, suggesting that large numbers of these channels are needed to allow for significant whole-cell current activity, and providing a mechanism to reduce noise in the visual system.
Assuntos
Biofísica/métodos , Sequência de Aminoácidos , Animais , Canais de Cálcio/química , Canais de Cálcio Tipo L/química , Linhagem Celular Tumoral , Clonagem Molecular , DNA/química , DNA Complementar/metabolismo , Eletrofisiologia , Ácido Glutâmico/química , Humanos , Cinética , Dados de Sequência Molecular , Probabilidade , Estrutura Terciária de Proteína , RNA/química , Ratos , Homologia de Sequência de Aminoácidos , Fatores de Tempo , TransfecçãoRESUMO
In humans, three isoforms of the T-type (Ca(v)3.1) calcium-channel alpha(1) subunit have been reported as a result of alternate splicing of exons 25 and 26 in the III-IV linker region (Ca(v)3.1a, Ca(v)3.1b or Ca(v)3.1bc). In the present study, we report that human glioma express Ca(v)3.1 channels in situ, that splicing of these exons is uniquely regulated and that there is expression of a glioma-specific novel T-type variant (Ca(v)3.1ac). 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 Ca(v)3.1 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, Ca(v)3.1a transcripts predominate, while Ca(v)3.1b is mostly fetal-specific. RT-PCR results on glioma and glioma cell lines showed that Ca(v)3.1 expression in tumor cells resemble fetal brain expression pattern as Ca(v)3.1bc is predominantly expressed. In addition, we identified a novel splice variant, Ca(v)3.1ac, 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 Ca(v)3.1ac displays similar current-voltage and steady-state inactivation properties compared with Ca(v)3.1b, but a slower recovery from inactivation. Taken together, our data suggest glioma-specific Ca(v)3.1 gene regulation, which could possibly contribute to tumor pathogenesis.