RESUMEN
BACKGROUND: KCNH2 gene mutations disrupting rapid component of I(K) (I(Kr)) underlie type 2 congenital long QT syndrome (LQT2). Startled auditory stimuli are specific symptomatic triggers in LQT2, thus suggesting fast arrhythmogenic mechanism. OBJECTIVE: We investigated acute alpha(1A)- and cyclic adenosine monophosphate (cAMP)-related beta-adrenergic modulation of I(Kr) in HL-1 cardiomyocytes, wild type (WT)- and 2 LQT2-associated mutant Kv11.1 channels (Y43D- and K595E-Kv11.1) reconstituted in Chinese hamster ovary (CHO) cells. METHODS: I(Kr) and Kv11.1 currents were recorded using the whole-cell patch-clamp technique and confocal microscopy of HL-1 cardiomyocytes transfected with green fluorescent protein (GFP)-tagged pleckstrin homology domain of phospholipase C-delta(1) visualized fluctuations of membrane phosphatidylinositol 4,5-bisphosphate (PIP(2)) content. RESULTS: In HL-1 cardiomyocytes expressing human alpha(1A)-adrenoceptor, superfusion with phenylephrine significantly reduced I(Kr) amplitude, shifted current activation to more positive potentials, and accelerated kinetics of deactivation. Confocal images showed a decline of membrane PIP(2) content during phenylephrine exposure. Simultaneous application of adenylyl cyclase activator forskolin and phosphodiesterase inhibitor 3-isobutyl-1-methylxantine (IBMX) shifted I(Kr) activation to more negative potentials and decreased tail current amplitudes after depolarizations between +10 and +50 mV. In CHO cells, alpha(1A)-adrenoceptor activation downregulated WT-Kv11.1 channels and forskolin/IBMX produced a dual effect. Expressed alone, the Y43D-Kv11.1 or K595E-Kv11.1 channel had no measurable function. However, co-expression of WT-Kv11.1 and each mutant protein evoked currents with loss-of-function alterations but identical to WT-Kv11.1 alpha(1A)- and forskolin/IBMX-induced regulation. CONCLUSION: Acute adrenergic regulation of at least 2 Kv11.1 mutant channels is preserved as in WT-Kv11.1 and native I(Kr). Suppression of alpha(1A)-adrenoceptor-related transduction might have therapeutic implications in some cases of LQT2.
Asunto(s)
Síndrome de QT Prolongado/fisiopatología , Miocitos Cardíacos/fisiología , Canales de Potasio con Entrada de Voltaje/fisiología , Receptores Adrenérgicos alfa/fisiología , Receptores Adrenérgicos beta/fisiología , Adulto , Animales , Células Cultivadas , Cricetinae , Modelos Animales de Enfermedad , Técnicas Electrofisiológicas Cardíacas , Femenino , Humanos , Síndrome de QT Prolongado/congénito , Síndrome de QT Prolongado/genética , Microscopía Confocal , Técnicas de Placa-Clamp , Canales de Potasio con Entrada de Voltaje/genéticaRESUMEN
BACKGROUND: Long QT syndromes (LQTS) are inherited diseases involving mutations to genes encoding a number of cardiac ion channels and a membrane adaptor protein. The MinK protein is a cardiac K-channel accessory subunit encoded by the KCNE1 gene, mutations of which are associated with the LQT5 form of LQTS. OBJECTIVE: The purpose of this study was to search for the KCNE1 mutations and clarify the function of those mutations. METHODS: We conducted a genetic screen of KCNE1 mutations in 151 Japanese LQTS patients using the denaturing high-performance liquid chromatography-WAVE system and direct sequencing. In two LQTS patients, we identified two KCNE1 missense mutations, located in the MinK N- and C-terminal domains. The functional effects of these mutations were examined by heterologous coexpression with KCNQ1 and KCNH2. RESULTS: One mutation, which was identified in a 67-year-old woman, A8V, was novel. Her electrocardiogram (ECG) revealed marked bradycardia and QT interval prolongation. Another mutation, R98W, was identified in a 19-year-old woman. She experienced syncope followed by palpitation in exercise. At rest, her ECG showed bradycardia with mild QT prolongation, which became more prominent during exercise. In electrophysiological analyses, R98W produced reduced I(Ks) currents with a positive shift in the half activation voltages. In addition, when the A8V mutation was coexpressed with KCNH2, this reduced current magnitude, which is suggestive of a modifier effect by the A8V KCNE1 mutation on I(Kr). CONCLUSION: KCNE1 mutations may be associated with mild LQTS phenotypes, and KCNE1 gene screening is of clinical importance for asymptomatic and mild LQTS patients.