RESUMEN
The voltage-gated sodium ion channel NaV1.7 is crucial in pain signaling. We examined how auxiliary ß2 and ß3 subunits and the phosphorylation state of the channel influence its biophysical properties and pharmacology. The human NaV1.7α subunit was co-expressed with either ß2 or ß3 subunits in HEK-293 cells. The ß2 subunits and the NaV1.7α, however, were barely associated as evidenced by immunoprecipitation. Therefore, the ß2 subunits did not change the biophysical properties of the channel. In contrast, ß3 subunit was clearly associated with NaV1.7α. This subunit had a significant degree of glycosylation, and only the fully glycosylated ß3 subunit was associated with the NaV1.7α. Electrophysiological characterisation revealed that the ß3 subunit had small but consistent effects: a right-hand shift of the steady-state inactivation and faster recovery from inactivation. Furthermore, the ß3 subunit reduced the susceptibility of NaV1.7α to several sodium channel blockers. In addition, we assessed the functional effect of NaV1.7α phosphorylation. Inhibition of kinase activity increased channel inactivation, while the blocking phosphatases produced the opposite effect. In conclusion, co-expression of ß subunits with NaV1.7α, to better mimic the native channel properties, may be ineffective in cases when subunits are not associated, as shown in our experiments with ß2. The ß3 subunit significantly influences the function of NaV1.7α and, together with the phosphorylation of the channel, regulates its biophysical and pharmacological properties. These are important findings to take into account when considering the role of NaV1.7 channel in pain signaling.
Asunto(s)
Activación del Canal Iónico , Canal de Sodio Activado por Voltaje NAV1.7/metabolismo , Procesamiento Proteico-Postraduccional , Glicosilación , Células HEK293 , Humanos , Canal de Sodio Activado por Voltaje NAV1.7/genética , Fosforilación , Multimerización de Proteína , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , Bloqueadores de los Canales de Sodio/farmacologíaRESUMEN
High blood pressure is the leading risk factor for death worldwide. One of the hallmarks is a rise of peripheral vascular resistance, which largely depends on arteriole tone. Ca2+-activated chloride currents (CaCCs) in vascular smooth muscle cells (VSMCs) are candidates for increasing vascular contractility. We analyzed the vascular tree and identified substantial CaCCs in VSMCs of the aorta and carotid arteries. CaCCs were small or absent in VSMCs of medium-sized vessels such as mesenteric arteries and larger retinal arterioles. In small vessels of the retina, brain, and skeletal muscle, where contractile intermediate cells or pericytes gradually replace VSMCs, CaCCs were particularly large. Targeted disruption of the calcium-activated chloride channel TMEM16A, also known as ANO1, in VSMCs, intermediate cells, and pericytes eliminated CaCCs in all vessels studied. Mice lacking vascular TMEM16A had lower systemic blood pressure and a decreased hypertensive response following vasoconstrictor treatment. There was no difference in contractility of medium-sized mesenteric arteries; however, responsiveness of the aorta and small retinal arterioles to the vasoconstriction-inducing drug U46619 was reduced. TMEM16A also was required for peripheral blood vessel contractility, as the response to U46619 was attenuated in isolated perfused hind limbs from mutant mice. Out data suggest that TMEM16A plays a general role in arteriolar and capillary blood flow and is a promising target for the treatment of hypertension.
Asunto(s)
Presión Sanguínea/efectos de los fármacos , Canales de Cloruro/metabolismo , Hipertensión/fisiopatología , Ácido 15-Hidroxi-11 alfa,9 alfa-(epoximetano)prosta-5,13-dienoico/farmacología , Animales , Anoctamina-1 , Arteriolas/patología , Presión Sanguínea/fisiología , Encéfalo/metabolismo , Clonación Molecular , ADN Complementario/metabolismo , Electrofisiología , Antagonistas de Estrógenos/farmacología , Células HEK293 , Humanos , Hipertensión/tratamiento farmacológico , Potenciales de la Membrana/efectos de los fármacos , Arterias Mesentéricas/patología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Músculo Liso Vascular/citología , Proteínas de Neoplasias/metabolismo , Pericitos/metabolismo , Retina/metabolismo , Tamoxifeno/farmacología , Factores de Tiempo , Resistencia Vascular , Vasoconstrictores/farmacologíaRESUMEN
At least five subtypes of voltage-gated (Kv1) channels occur in neurons as tetrameric combinations of different alpha subunits. Their involvement in controlling cell excitability and synaptic transmission make them potential targets for neurotherapeutics. As a prerequisite for this, we established herein how the characteristics of hetero-oligomeric K(+) channels can be influenced by alpha subunit composition. Since the three most prevalent Kv1 subunits in brain are Kv1.2, 1.1 and 1.6, new Kv1.6-1.2 and Kv1.1-1.2 concatenated constructs in pIRES-EGFP were stably expressed in HEK cells and the biophysical plus pharmacological properties of their K(+) currents determined relative to those for the requisite homo-tetramers. These heteromers yielded delayed-rectifier type K(+) currents whose activation, deactivation and inactivation parameters are fairly similar although substituting Kv1.1 with Kv1.6 led to a small negative shift in the conductance-voltage relationship, a direction unexpected from the characteristics of the parental homo-tetramers. Changes resulting from swapping Kv1.6 for Kv1.1 in the concatemers were clearly discerned with two pharmacological agents, as measured by inhibition of the K(+) currents and Rb(+) efflux. alphaDendrotoxin and 4-aminopyridine gave a similar blockade of both hetero-tetramers, as expected. Most important for pharmacological dissection of channel subtypes, dendrotoxin(k) and tetraethylammonium readily distinguished the susceptible Kv1.1-1.2 containing oligomers from the resistant Kv1.6-1.2 channels. Moreover, the discriminating ability of dendrotoxin(k) was further confirmed by its far greater ability to displace (125)I-labelled alphadendrotoxin binding to Kv1.1-1.2 than Kv1.6-1.2 channels. Thus, due to the profiles of these two channel subtypes being found to differ, it seems that only multimers corresponding to those present in the nervous system provide meaningful targets for drug development.
Asunto(s)
Canal de Potasio Kv.1.1/fisiología , Canales de Potasio/metabolismo , Potasio/metabolismo , Línea Celular Transformada , Relación Dosis-Respuesta a Droga , Relación Dosis-Respuesta en la Radiación , Interacciones Farmacológicas , Estimulación Eléctrica , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Canal de Potasio Kv.1.1/química , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/fisiología , Técnicas de Placa-Clamp , Péptidos/farmacología , Potasio/farmacología , Bloqueadores de los Canales de Potasio/farmacología , Canales de Potasio/clasificación , Canales de Potasio/genética , Estructura Terciaria de Proteína , Transfección/métodosRESUMEN
Whole cell recordings of excitatory postsynaptic potentials/currents (EPSPs/EPSCs) evoked by minimal stimulation of commissural-associative (CF) and mossy fibre (MF) inputs were performed in CA3 pyramidal neurons. Paired responses (at 50 ms intervals) were recorded before, during and after hyperpolarization of the postsynaptic membrane (20-30 mV for 15-35 min). Membrane hyperpolarization produced a supralinear increase of EPSPs/EPSCs amplitude in MF-inputs. Synaptic responses remained potentiated for the rest of the recording period (up to 40 min) after resetting the membrane potential to control level (221 +/- 60%, n = 15 and 219 +/- 61%, n = 11 for MF-EPSP and MF-EPSC, respectively). We shall refer to this effect as hyperpolarization-induced LTP (HI-LTP). In the absence of afferent stimulation, membrane hyperpolarization was unable to produce HI-LTP. In contrast to MF-EPSPs, the mean amplitude of CF-EPSPs did not increase significantly after hyperpolarization relative to controls (138 +/- 29%, n = 22). HI-LTP was associated with modifications of classical indices of presynaptic release: paired-pulse facilitation, failures rate, coefficient of variation of EPSP amplitudes and quantal content. The induction of HI-LTP was NMDA independent but was dependent on metabotropic glutamate receptors (mGluRs) activation and calcium release from inositol 1,4,5-triphosphate (IP3)-sensitive intracellular stores: it was prevented by mGluR antagonist, intracellular heparin and BAPTA. We conclude that while the induction of HI-LTP was postsynaptic, its expression was presynaptic.