RESUMO
Cardiac L-type voltage-dependent Ca(2+) channels are heteromultimeric polypeptide complexes of alpha(1)-, alpha(2)/delta-, and beta-subunits. The alpha(2)/delta-1-subunit possesses a stereoselective, high-affinity binding site for gabapentin, widely used to treat epilepsy and postherpetic neuralgic pain as well as sleep disorders. Mutations in alpha(2)/delta-subunits of voltage-dependent Ca(2+) channels have been associated with different diseases, including epilepsy. Multiple heterologous coexpression systems have been used to study the effects of the deletion of the alpha(2)/delta-1-subunit, but attempts at a conventional knockout animal model have been ineffective. We report the development of a viable conventional knockout mouse using a construct targeting exon 2 of alpha(2)/delta-1. While the deletion of the subunit is not lethal, these animals lack high-affinity gabapentin binding sites and demonstrate a significantly decreased basal myocardial contractility and relaxation and a decreased L-type Ca(2+) current peak current amplitude. This is a novel model for studying the function of the alpha(2)/delta-1-subunit and will be of importance in the development of new pharmacological therapies.
Assuntos
Canais de Cálcio Tipo L/fisiologia , Canais de Cálcio/fisiologia , Aminas/metabolismo , Animais , Sítios de Ligação/efeitos dos fármacos , Sítios de Ligação/genética , Western Blotting , Canais de Cálcio/efeitos dos fármacos , Canais de Cálcio/genética , Canais de Cálcio Tipo L/efeitos dos fármacos , Canais de Cálcio Tipo L/genética , Ácidos Cicloexanocarboxílicos/metabolismo , Eletrofisiologia , Éxons/genética , Gabapentina , Genótipo , Coração/efeitos dos fármacos , Camundongos , Camundongos Knockout , Músculo Esquelético/citologia , Músculo Esquelético/metabolismo , Contração Miocárdica/efeitos dos fármacos , Contração Miocárdica/genética , Miócitos Cardíacos/efeitos dos fármacos , Técnicas de Patch-Clamp , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Ácido gama-Aminobutírico/metabolismoRESUMO
Voltage-gated L-type Ca(2+) channels (LCCs) provide Ca(2+) ingress into cardiac myocytes and play a key role in intracellular Ca(2+) homeostasis and excitation-contraction coupling. We investigated the effects of a constitutive increase of LCC density on Ca(2+) signaling in ventricular myocytes from 4-month-old transgenic (Tg) mice overexpressing the alpha(1) subunit of LCC in the heart. At this age, cells were somewhat hypertrophic as reflected by a 20% increase in cell capacitance relative to those from nontransgenic (Ntg) littermates. Whole cell I(Ca) density in Tg myocytes was elevated by 48% at 0 mV compared with the Ntg group. Single-channel analysis detected an increase in LCC density with similar conductance and gating properties. Although the overexpressed LCCs triggered an augmented SR Ca(2+) release, the "gain" function of EC coupling was uncompromised, and SR Ca(2+) content, diastolic cytosolic Ca(2+), and unitary properties of Ca(2+) sparks were unchanged. Importantly, the enhanced I(Ca) entry and SR Ca(2+) release were associated with an upregulation of the Na(+)-Ca(2+) exchange activity (indexed by the half decay time of caffeine-elicited Ca(2+) transient) by 27% and SR Ca(2+) recycling by approximately 35%. Western analysis detected a 53% increase in the Na(+)-Ca(2+) exchanger expression but no change in the abundance of ryanodine receptor (RyR), SERCA2, and phospholamban. Analysis of I(Ca) kinetics suggested that SR Ca(2+) release-dependent inactivation of LCCs remains intact in Tg cells. Thus, in spite of the modest cardiac hypertrophy, the overexpressed LCCs form functional coupling with RyRs, preserving both orthograde and retrograde Ca(2+) signaling between LCCs and RyRs. These results also suggest that a modest but sustained increase in Ca(2+) influx triggers a coordinated remodeling of Ca(2+) handling to maintain Ca(2+) homeostasis.