In the congenital long-QT syndrome, prolongation of the cardiac action potential occurs by an unknown mechanism and predisposes individuals to syncope and sudden death as a result of ventricular arrhythmias. Genetic heterogeneity has been demonstrated for autosomal dominant long-QT syndrome by the identification of multiple distinct loci, and associated mutations in two candidate genes have recently been reported. One form of hereditary long QT (LQT3) has been linked to a mutation in the gene encoding the human heart voltage-gated sodium-channel alpha-subunit (SCN5A on chromosome 3p21). Here we characterize this mutation using heterologous expression of recombinant human heart sodium channels. Mutant channels show a sustained inward current during membrane depolarization. Single-channel recordings indicate that mutant channels fluctuate between normal and non-inactivating gating modes. Persistent inward sodium current explains prolongation of cardiac action potentials, and provides a molecular mechanism for this form of congenital long-QT syndrome.

The congenital long QT syndrome (LQTS) is an inherited disorder characterized by a prolonged cardiac action potential. This delay in cellular repolarization can lead to potentially fatal arrhythmias. One form of LQTS (LQT3) has been linked to the human cardiac voltage-gated sodium channel gene (SCN5A). Three distinct mutations have been identified in the sodium channel gene. The biophysical and functional characteristics of each of these mutant channels were determined by heterologous expression of a recombinant human heart sodium channel in a mammalian cell line. Each mutation caused a sustained, non-inactivating sodium current amounting to a few percent of the peak inward sodium current, observable during long (>50 msec) depolarizations. The voltage dependence and rate of inactivation were altered, and the rate of recovery from inactivation was changed compared with wild-type channels. These mutations in diverse regions of the ion channel protein, all produced a common defect in channel gating that can cause the long QT phenotype. The sustained inward current caused by these mutations will prolong the action potential. Furthermore, they may create conditions that promote arrhythmias due to prolonged depolarization and the altered recovery from inactivation. These results provide insights for successful intervention in the disease.

The congenital long QT syndrome (LQTS) is an inherited disorder characterized by a delay in cardiac cellular repolarization leading to cardiac arrhythmias and sudden death often in young people. One form of the disease (

SUMMARY1. Receptor-mediated modulation of the delayed outward potassium current (IK) was investigated in guinea-pig single ventricular cells by using whole-cell voltage clamp and intracellular dialysis.2. Isoprenaline increased IK in a dose-dependent manner with a half-maximum dose of 1-8 x 10-8 M. Isoprenaline (10-6 M) maximally increased IK by a factor of 2-85. This effect did not depend on the concentration of intracellular Ca2+ ([Ca2+]1).3. External application of 10-5 M-forskolin and internal application of 5 x 10-5 Mcyclic AMP or 5 x 10-6 M of the catalytic subunit of cyclic AMP-dependent protein kinase (PKA) also increased IK about 3-fold. The effect of isoprenaline on IK was masked by previous application of cyclic AMP.4. All the above phosphorylating agents increased the amplitude of IK without a significant change in the current kinetics.5. In the presence of 10-5 M-forskolin, an additional application of 10-8 M-12-Otetradecanoylphorbol-13-acetate, an activator of protein kinase C (PKC), produced a further increase in IK, suggesting that the active sites of PKA and PKC on the IK channel are different.6. Acetylcholine (10-6 M) suppressed IK when the current was previously enhanced by 2 x 10-8 M-isoprenaline, but had little effect in the absence of isoprenaline.7. We conclude that f8-adrenergic modulation of IK is mediated by cyclic AMPdependent phosphorylation but not by an increase in [Ca2+]1, that PKA and PKC enhance IK independently, and that acetylcholine antagonizes f,-adrenergic stimulation ofIK most probably by inhibiting adenylate cyclase.

The role of adenosine triphosphate (ATP) in the regulation of L-type Ca2+ channel activity was investigated in inside-out patches from guinea-pig ventricular cells, in which the Ca2+ channel activity had been reprimed by application of cytoplasm from bovine heart. Passing the cytoplasm through a diethylaminoethyl (DEAE)-sepharose column or heating at 60 degrees C for 20 min attenuated the induction Ca2+ channel activity to 6-13% of that in the preceding cell-attached patch. Addition of 10 mM MgATP to the cytoplasm greatly improved the potency of cytoplasm in restoring Ca2+ channel activity (to 83 +/- 22%, mean +/- SE). This effect of MgATP was also produced, although with lower potency, by K2ATP (61 +/- 20%) or 5'-adenylylimidodiphosphate (AMP-PNP, 39 +/- 7%), a non-hydrolyzable ATP analogue, suggesting that hydrolysis of ATP is not required for the stimulatory effect on channel activity. A non-specific protein kinase inhibitor H8 (50-100 microM) did not inhibit the effect of cytoplasm + MgATP on channel activity, suggesting the involvement of a pathway independent of phosphorylation. We conclude that ATP regulates Ca2+ channel activity in dual pathways: one with, and the other without, protein phosphorylation.