Novel arrhythmogenic mechanism revealed by a long-QT syndrome mutation in the cardiac Na(+) channel.

Variant 3 of the congenital long-QT syndrome (LQTS-3) is caused by mutations in the gene encoding the alpha subunit of the cardiac Na(+) channel. In the present study, we report a novel LQTS-3 mutation, E1295K (EK), and describe its functional consequences when expressed in HEK293 cells. The clinical phenotype of the proband indicated QT interval prolongation in the absence of T-wave morphological abnormalities and a steep QT/R-R relationship, consistent with an LQTS-3 lesion. However, biophysical analysis of mutant channels indicates that the EK mutation changes channel activity in a manner that is distinct from previously investigated LQTS-3 mutations. The EK mutation causes significant positive shifts in the half-maximal voltage (V(1/2)) of steady-state inactivation and activation (+5.2 and +3.4 mV, respectively). These gating changes shift the window of voltages over which Na(+) channels do not completely inactivate without altering the magnitude of these currents. The change in voltage dependence of window currents suggests that this alteration in the voltage dependence of Na(+) channel gating may cause marked changes in action potential duration because of the unique voltage-dependent rectifying properties of cardiac K(+) channels that underlie the plateau and terminal repolarization phases of the action potential. Na(+) channel window current is likely to have a greater effect on net membrane current at more positive potentials (EK channels) where total K(+) channel conductance is low than at more negative potentials (wild-type channels), where total K(+) channel conductance is high. These findings suggest a fundamentally distinct mechanism of arrhythmogenesis for congenital LQTS-3.

TEA(+)-sensitive KCNQ1 constructs reveal pore-independent access to KCNE1 in assembled I(Ks) channels.

I(Ks), a slowly activating delayed rectifier K(+) current through channels formed by the assembly of two subunits KCNQ1 (KvLQT1) and KCNE1 (minK), contributes to the control of the cardiac action potential duration. Coassembly of the two subunits is essential in producing the characteristic and physiologically critical kinetics of assembled channels, but it is not yet clear where or how these subunits interact. Previous investigations of external access to the KCNE1 protein in assembled I(Ks) channels relied on occlusion of the pore by extracellular application of TEA(+), despite the very low TEA(+) sensitivity (estimated EC(50) > 100 mM) of channels encoded by coassembly of wild-type KCNQ1 with the wild type (WT) or a series of cysteine-mutated KCNE1 constructs. We have engineered a high affinity TEA(+) binding site into the h-KCNQ1 channel by either a single (V319Y) or double (K318I, V319Y) mutation, and retested it for pore-delimited access to specific sites on coassembled KCNE1 subunits. Coexpression of either KCNQ1 construct with WT KCNE1 in Chinese hamster ovary cells does not alter the TEA(+) sensitivity of the homomeric channels (IC(50) approximately 0.4 mM [TEA(+)](out)), providing evidence that KCNE1 coassembly does not markedly alter the structure of the outer pore of the KCNQ1 channel. Coexpression of a cysteine-substituted KCNE1 (F54C) with V319Y significantly increases the sensitivity of channels to external Cd(2+), but neither the extent of nor the kinetics of the onset of (or the recovery from) Cd(2+) block was affected by [TEA(+)](o) at 10x the IC(50) for channel block. These data strongly suggest that access of Cd(2+) to the cysteine-mutated site on KCNE1 is independent of pore occlusion caused by TEA(+) binding to the outer region of the KCNE1/V319Y pore, and that KCNE1 does not reside within the pore region of the assembled channels.

A region in IVS5 of the human cardiac L-type calcium channel is required for the use-dependent block by phenylalkylamines and benzothiazepines.

Mutations in motif IVS5 and IVS6 of the human cardiac calcium channel were made using homologous residues from the rat brain sodium channel 2a. [3H]PN200-110 and allosteric binding assays revealed that the dihydropyridine and benzothiazepine receptor sites maintained normal coupling in the chimeric mutant channels. Whole cell voltage clamp recording from Xenopus oocytes showed a dramatically slowed inactivation and a complete loss of use-dependent block for mutations in the cytoplasmic connecting link to IVS5 (HHT-5371) and in IVS5 transmembrane segment (HHT-5411) with both diltiazem and verapamil. However, the use-dependent block by isradipine was retained by these two mutants. For mutants HHT-5411 and HHT-5371, the residual current appeared associated with a loss of voltage dependence in the rate of inactivation indicating a destabilization of the inactivated state. Furthermore, both HHT-5371 and -5411 recovered from inactivation significantly faster after drug block than that of the wild type channel. Our data demonstrate that accelerated recovery of HHT-5371 and HHT-5411 decreased accumulation of these channels in inactivation during pulse trains and suggest a close link between inactivation gating of the channel and use-dependent block by phenylalkylamines and benzothiazepines and provide evidence of a role for the transmembrane and cytoplasmic regions of IVS5 in the use-dependent block by diltiazem and verapamil.

Location of ryanodine receptor binding site on skeletal muscle triadin.

The binding between intact triadin or expressed triadin peptides and the ryanodine receptor has been investigated using membrane overlay and affinity chromatography. Ryanodine receptor binds to triadin blotted onto nitrocellulose with a KD of 40 nM in a medium containing 150 mM NaCl. The binding is substantially inhibited by hypertonic salt solution. Blot overlay experiments show that ryanodine receptor binds to bacterially expressed peptides, triadin(110-280), triadin(110-267), and triadin(279-674), but to no other moieties of the protein (numbers in parentheses are the residue positions). This binding is strongly inhibited by hypertonic salt solution. The same three triadin peptides as well as triadin(68-267), when attached to a glutathione column, bind to the ryanodine receptor. However, triadin(110-280) binds with high affinity, while triadin(68-267), triadin(110-267), and triadin(279-674) bind with low affinity. Triadin(258-280), triadin(267-280), and triadin(258-299) all bind to the ryanodine receptor with high affinity. On the other hand, a construct containing triadin(267-280), but preceded by nine residues of heterologous amino acids, does not bind significantly. These observations indicate two types of binding between triadin and the ryanodine receptor: (1) a low-affinity ionic interaction of large portions of triadin; (2) a specific high-affinity binding of a short relatively hydrophobic segment. The binding of this segment is probably the physiologically important domain for attachment between triadin and the ryanodine receptor.

Extraction of junctional complexes from triad junctions of rabbit skeletal muscle.

Triadin in skeletal muscle exists as a disulfide linked oligomer. It does not dissolve well in CHAPS detergent even in 1 M KCl, but is solubilized after reduction to its monomer by the addition of 2-mercaptoethanol. Purified reduced triadin is not retained on a hydroxylapatite column in the presence of 30 mM Potassium phosphate, while the junctional foot protein and dihydropyridine receptor purified in the absence of triadin are both retained. In contrast, triadin solubilized as a detergent extract of reduced triadic vesicles is retained by the hydroxylapatite column and elutes concomitantly with the junctional foot protein and dihydropyridine receptor. These findings contrast with the observation that native non-reduced triadin is tightly bound to hydroxylapatite and can be separated from the dihydropyridine receptor and the junctional foot protein with elevated potassium phosphate concentrations. Triadin derived from a detergent extract of reduced vesicles is retained with the hydroxytapatite column in the presence of 180 mM potassium phosphate (0 KCl) which eluted a portion of the junctional foot protein and dihydropyridine receptor. Triadin can then be eluted with the remaining portion of junctional foot protein and dihydropyridine receptor upon the addition of KCl (820 mM) to the 180 mM potassium phosphate medium. Gel electrophoresis confirmed the enrichment of junctional proteins in the 180 mM KPi/820 mM KCl eluate. Rate zonal centrifugation of the 180 mM KPi/820 mM KCl eluate shows that a portion of triadin co-migrates with the dihydropyridine receptor indicative of a much higher molecular weight entity than monomeric triadin. Triadin and the dihydropyridine receptor were, however, separated from the junctional foot protein on rate zonal centrifugation. The dissociated proteins of the complex elute from hydroxylapatite columns similar to the purified proteins. Triadin in the high salt hydroxylapatite extract could also be immunoprecipitated by a monoclonal antibody to the junctional foot protein. Furthermore, the dihydropyridine receptor is immunoprecipitated by a monoclonal antibody directed against triadin providing another indication of a complex between the three proteins. Collectively, these results demonstrate a role for triadin as the linkage between the junctional foot protein and dihydropyridine receptor creating a ternary complex at the triad junction in skeletal muscle.