RESUMEN
The auxiliary α2δ subunits of voltage-gated calcium (CaV) channels are key to augmenting expression and function of CaV1 and CaV2 channels, and are also important drug targets in several therapeutic areas, including neuropathic pain. The α2δ proteins are translated as preproteins encoding both α2 and δ, and post-translationally proteolyzed into α2 and δ subunits, which remain associated as a complex. In this study, we have identified ADAM17 as a key protease involved in proteolytic processing of pro-α2δ-1 and α2δ-3 subunits. We provide three lines of evidence: First, proteolytic cleavage is inhibited by chemical inhibitors of particular metalloproteases, including ADAM17. Second, proteolytic cleavage of both α2δ-1 and α2δ-3 is markedly reduced in cell lines by knockout of ADAM17 but not ADAM10. Third, proteolytic cleavage is reduced by the N-terminal active domain of TIMP-3 (N-TIMP-3), which selectively inhibits ADAM17. We have found previously that proteolytic cleavage into mature α2δ is essential for the enhancement of CaV function, and in agreement, knockout of ADAM17 inhibited the ability of α2δ-1 to enhance both CaV2.2 and CaV1.2 calcium currents. Finally, our data also indicate that the main site of proteolytic cleavage of α2δ-1 is the Golgi apparatus, although cleavage may also occur at the plasma membrane. Thus, our study identifies ADAM17 as a key protease required for proteolytic maturation of α2δ-1 and α2δ-3, and thus a potential drug target in neuropathic pain.
Asunto(s)
Neuralgia , Inhibidor Tisular de Metaloproteinasa-3 , Humanos , Inhibidor Tisular de Metaloproteinasa-3/metabolismo , Canales de Calcio Tipo N/genética , Proteolisis , Calcio de la Dieta/metabolismo , Péptido Hidrolasas/metabolismo , Proteína ADAM17/genéticaRESUMEN
Patients with amyotrophic lateral sclerosis (ALS) often show hallmarks of type 2 diabetes mellitus (T2DM). However, the causal link between ALS and T2DM has remained a mystery. We now demonstrate that 60% of ALS patients with T2DM (ALS-T2DM) have sera that exaggerated K+-induced increases in cytosolic free Ca2+ concentration ([Ca2+]i) in mouse islet cells. The effect was attributed to the presence of pathogenic immunoglobulin Gs (IgGs) in ALS-T2DM sera. The pathogenic IgGs immunocaptured the voltage-dependent Ca2+ (CaV) channel subunit CaVα2δ1 in the plasma membrane enhancing CaV1 channel-mediated Ca2+ influx and [Ca2+]i, resulting in impaired mitochondrial function. Consequently, impairments in [Ca2+]i dynamics, insulin secretion, and cell viability occurred. These data reveal that patients with ALS-T2DM carry cytotoxic ALS-T2DM-IgG autoantibodies that serve as a causal link between ALS and T2DM by immunoattacking CaVα2δ1 subunits. Our findings may lay the foundation for a pharmacological treatment strategy for patients suffering from a combination of these diseases.
RESUMEN
The α2δ-1 subunit of voltage-gated calcium channels is upregulated after sensory nerve injury and is also the therapeutic target of gabapentinoid drugs. It is therefore likely to play a key role in the development of neuropathic pain. In this study, we have examined mice in which α2δ-1 gene expression is disrupted, to determine whether α2δ-1 is involved in various modalities of nociception, and for the development of behavioral hypersensitivity after partial sciatic nerve ligation (PSNL). We find that naive α2δ-1(-/-) mice show a marked behavioral deficit in mechanical and cold sensitivity, but no change in thermal nociception threshold. The lower mechanical sensitivity is mirrored by a reduced in vivo electrophysiological response of dorsal horn wide dynamic range neurons. The CaV2.2 level is reduced in brain and spinal cord synaptosomes from α2δ-1(-/-) mice, and α2δ-1(-/-) DRG neurons exhibit lower calcium channel current density. Furthermore, a significantly smaller number of DRG neurons respond to the TRPM8 agonist menthol. After PSNL, α2δ-1(-/-) mice show delayed mechanical hypersensitivity, which only develops at 11 d after surgery, whereas in wild-type littermates it is maximal at the earliest time point measured (3 d). There is no compensatory upregulation of α2δ-2 or α2δ-3 after PSNL in α2δ-1(-/-) mice, and other transcripts, including neuropeptide Y and activating transcription factor-3, are upregulated normally. Furthermore, the ability of pregabalin to alleviate mechanical hypersensitivity is lost in PSNL α2δ-1(-/-) mice. Thus, α2δ-1 is essential for rapid development of mechanical hypersensitivity in a nerve injury model of neuropathic pain.
Asunto(s)
Canales de Calcio/genética , Hiperalgesia/genética , Neuralgia/genética , Traumatismos de los Nervios Periféricos/genética , Células Receptoras Sensoriales/fisiología , Animales , Canales de Calcio/metabolismo , Frío , Ganglios Espinales/metabolismo , Ganglios Espinales/fisiopatología , Hiperalgesia/metabolismo , Hiperalgesia/fisiopatología , Ratones , Ratones Noqueados , Neuralgia/metabolismo , Neuralgia/fisiopatología , Umbral del Dolor/fisiología , Traumatismos de los Nervios Periféricos/metabolismo , Traumatismos de los Nervios Periféricos/fisiopatología , Estimulación Física , Nervio Ciático/lesiones , Nervio Ciático/metabolismo , Nervio Ciático/fisiopatologíaRESUMEN
At least 5% of individuals with hypertension have adrenal aldosterone-producing adenomas (APAs). Gain-of-function mutations in KCNJ5 and apparent loss-of-function mutations in ATP1A1 and ATP2A3 were reported to occur in APAs. We find that KCNJ5 mutations are common in APAs resembling cortisol-secreting cells of the adrenal zona fasciculata but are absent in a subset of APAs resembling the aldosterone-secreting cells of the adrenal zona glomerulosa. We performed exome sequencing of ten zona glomerulosa-like APAs and identified nine with somatic mutations in either ATP1A1, encoding the Na(+)/K(+) ATPase α1 subunit, or CACNA1D, encoding Cav1.3. The ATP1A1 mutations all caused inward leak currents under physiological conditions, and the CACNA1D mutations induced a shift of voltage-dependent gating to more negative voltages, suppressed inactivation or increased currents. Many APAs with these mutations were <1 cm in diameter and had been overlooked on conventional adrenal imaging. Recognition of the distinct genotype and phenotype for this subset of APAs could facilitate diagnosis.
Asunto(s)
Enfermedades de la Corteza Suprarrenal/genética , Canales de Calcio Tipo L/genética , Hipertensión/genética , Mutación , ATPasa Intercambiadora de Sodio-Potasio/genética , Enfermedades de la Corteza Suprarrenal/complicaciones , Enfermedades de la Corteza Suprarrenal/diagnóstico , Sustitución de Aminoácidos , Canales de Calcio Tipo L/química , Canales de Calcio Tipo L/metabolismo , Análisis por Conglomerados , Femenino , Canales de Potasio Rectificados Internamente Asociados a la Proteína G/genética , Canales de Potasio Rectificados Internamente Asociados a la Proteína G/metabolismo , Perfilación de la Expresión Génica , Humanos , Hipertensión/diagnóstico , Hipertensión/etiología , Masculino , Conformación Proteica , ATPasa Intercambiadora de Sodio-Potasio/química , ATPasa Intercambiadora de Sodio-Potasio/metabolismoRESUMEN
The ß-subunits of voltage-gated calcium channels regulate their functional expression and properties. Two mechanisms have been proposed for this, an effect on gating and an enhancement of expression. With respect to the effect on expression, ß-subunits have been suggested to enhance trafficking by masking an unidentified endoplasmic reticulum (ER) retention signal. Here we have investigated whether, and how, ß-subunits affect the level of Ca(V)2.2 channels within somata and neurites of cultured sympathetic neurons. We have used YFP-Ca(V)2.2 containing a mutation (W391A), that prevents binding of ß-subunits to its I-II linker and found that expression of this channel was much reduced compared with WT CFP-Ca(V)2.2 when both were expressed in the same neuron. This effect was particularly evident in neurites and growth cones. The difference between the levels of YFP-Ca(V)2.2(W391A) and CFP-Ca(V)2.2(WT) was lost in the absence of co-expressed ß-subunits. Furthermore, the relative reduction of expression of Ca(V)2.2(W391A) compared with the WT channel was reversed by exposure to two proteasome inhibitors, MG132 and lactacystin, particularly in the somata. In further experiments in tsA-201 cells, we found that proteasome inhibition did not augment the cell surface Ca(V)2.2(W391A) level but resulted in the observation of increased ubiquitination, particularly of mutant channels. In contrast, we found no evidence for selective retention of Ca(V)2.2(W391A) in the ER, in either the soma or growth cones. In conclusion, there is a marked effect of ß-subunits on Ca(V)2.2 expression, particularly in neurites, but our results point to protection from proteasomal degradation rather than masking of an ER retention signal.
Asunto(s)
Canales de Calcio Tipo N/biosíntesis , Retículo Endoplásmico/metabolismo , Regulación de la Expresión Génica/fisiología , Proteínas del Tejido Nervioso/metabolismo , Neuritas/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Acetilcisteína/análogos & derivados , Acetilcisteína/farmacología , Sustitución de Aminoácidos , Animales , Células COS , Canales de Calcio Tipo N/genética , Chlorocebus aethiops , Inhibidores de Cisteína Proteinasa/farmacología , Retículo Endoplásmico/genética , Regulación de la Expresión Génica/efectos de los fármacos , Leupeptinas/farmacología , Mutación Missense , Proteínas del Tejido Nervioso/antagonistas & inhibidores , Proteínas del Tejido Nervioso/genética , Complejo de la Endopetidasa Proteasomal/genética , Inhibidores de Proteasoma , Conejos , RatasRESUMEN
Expression of the calcium channels Ca(V)2.1 and Ca(V)2.2 is markedly suppressed by co-expression with truncated constructs containing Domain I. This is the basis for the phenomenon of dominant negative suppression observed for many of the episodic ataxia type 2 mutations in Ca(V)2.1 that predict truncated channels. The process of dominant negative suppression has been shown previously to stem from interaction between the full-length and truncated channels and to result in downstream consequences of the unfolded protein response and endoplasmic reticulum-associated protein degradation. We have now identified the specific domain that triggers this effect. For both Ca(V)2.1 and Ca(V)2.2, the minimum construct producing suppression was the cytoplasmic N terminus. Suppression was enhanced by tethering the N terminus to the membrane with a CAAX motif. The 11-amino acid motif (including Arg(52) and Arg(54)) within the N terminus, which we have previously shown to be required for G protein modulation, is also essential for dominant negative suppression. Suppression is prevented by addition of an N-terminal tag (XFP) to the full-length and truncated constructs. We further show that suppression of Ca(V)2.2 currents by the N terminus-CAAX construct is accompanied by a reduction in Ca(V)2.2 protein level, and this is also prevented by mutation of Arg(52) and Arg(54) to Ala in the truncated construct. Taken together, our evidence indicates that both the extreme N terminus and the Arg(52), Arg(54) motif are involved in the processes underlying dominant negative suppression.