RESUMEN
Mutations in the KCNT1 potassium channel cause severe forms of epilepsy which are poorly controlled with current treatments. In vitro studies have shown that KCNT1-epilepsy mutations are gain of function, significantly increasing K+ current amplitudes. To investigate if Drosophila can be used to model human KCNT1 epilepsy, we generated Drosophila melanogaster lines carrying human KCNT1 with the patient mutation G288S, R398Q or R928C. Expression of each mutant channel in GABAergic neurons gave a seizure phenotype which responded either positively or negatively to 5 frontline epilepsy drugs most commonly administered to patients with KCNT1-epilepsy, often with little or no improvement of seizures. Cannabidiol showed the greatest reduction of the seizure phenotype while some drugs increased the seizure phenotype. Our study shows that Drosophila has the potential to model human KCNT1- epilepsy and can be used as a tool to assess new treatments for KCNT1- epilepsy.
Asunto(s)
Drosophila , Epilepsia , Canales de potasio activados por Sodio , Animales , Humanos , Drosophila/genética , Drosophila melanogaster/genética , Evaluación Preclínica de Medicamentos , Epilepsia/tratamiento farmacológico , Epilepsia/genética , Modelos Animales , Mutación , Proteínas del Tejido Nervioso/genética , Canales de potasio activados por Sodio/genética , Convulsiones/tratamiento farmacológico , Convulsiones/genética , TransgenesRESUMEN
The voltage-gated K+ channel plays a key role in atrial excitability, conducting the ultra-rapid rectifier K+ current (IKur) and contributing to the repolarization of the atrial action potential. In this study, we examine its regulation by hydrogen sulfide (H2S) in HL-1 cardiomyocytes and in HEK293 cells expressing human Kv1.5. Pacing induced remodeling resulted in shorting action potential duration, enhanced both Kv1.5 channel and H2S producing enzymes protein expression in HL-1 cardiomyocytes. H2S supplementation reduced these remodeling changes and restored action potential duration through inhibition of Kv1.5 channel. H2S also inhibited recombinant hKv1.5, lead to nitric oxide (NO) mediated S-nitrosylation and activated endothelial nitric oxide synthase (eNOS) by increased phosphorylation of Ser1177, prevention of NO formation precluded these effects. Regulation of Ikur by H2S has important cardiovascular implications and represents a novel and potential therapeutic target.
Asunto(s)
Fibrilación Atrial , Sulfuro de Hidrógeno , Canales de Potasio con Entrada de Voltaje , Humanos , Sulfuro de Hidrógeno/farmacología , Sulfuro de Hidrógeno/metabolismo , Fibrilación Atrial/metabolismo , Células HEK293 , Canal de Potasio Kv1.5/genética , Canal de Potasio Kv1.5/metabolismo , Miocitos Cardíacos/metabolismoRESUMEN
Renal tubular reabsorption is important for extracellular fluid homeostasis and much of this occurs via the receptor-mediated endocytic pathway. This pathway is disrupted in Dent's disease, an X-linked renal tubular disorder that is characterized by low-molecular-weight proteinuria, hypercalciuria, nephrolithiasis, and renal failure. Dent's disease is due to mutations of CLC-5, a chloride/proton antiporter, expressed in endosomes and apical membranes of renal tubules. Loss of CLC-5 function alters receptor-mediated endocytosis and trafficking of megalin and cubilin, although the underlying mechanisms remain to be elucidated. Here, we report that CLC-5 interacts with kinesin family member 3B (KIF3B), a heterotrimeric motor protein that facilitates fast anterograde translocation of membranous organelles. Using yeast two-hybrid, glutathione-S-transferase pull-down and coimmunoprecipitation assays, the COOH terminus of CLC-5 and the coiled-coil and globular domains of KIF3B were shown to interact. This was confirmed in vivo by endogenous coimmunoprecipitation of CLC-5 and KIF3B and codistribution with endosomal markers in mouse kidney fractions. Confocal live cell imaging in kidney cells further demonstrated association of CLC-5 and KIF3B, and transport of CLC-5-containing vesicles along KIF3B microtubules. KIF3B overexpression and underexpression, using siRNA, had reciprocal effects on whole cell chloride current amplitudes, CLC-5 cell surface expression, and endocytosis of albumin and transferrin. Clcn5(Y/-) mouse kidneys and isolated proximal tubular polarized cells showed increased KIF3B expression, whose effects on albumin endocytosis were dependent on CLC-5 expression. Thus, the CLC-5 and KIF3B interaction is important for CLC-5 plasma membrane expression and for facilitating endocytosis and microtubular transport in the kidney.