The function of the 12 positive charges in the 53-residue III/IV interdomain linker of the cardiac Na + channel is unclear. We have identified a four-generation family, including 17 gene carriers with long QT syndrome, Brugada syndrome, and conduction system disease with deletion of lysine 1500 (∆K1500) within the linker. Three family members died suddenly. We have examined the functional consequences of this mutation by measuring whole-cell and single-channel currents in 293-EBNA cells expressing the wild-type and ∆K1500 mutant channel. The mutation shifted the potential for half inactivation (V 1/2 h ∞ ) to more negative values and reduced its voltage dependence consistent with a reduction of inactivation valence of 1. The shift in inactivation was the result of an increase in closed-state inactivation rate (11-fold at -100 mV). The potential for half activation (V 1/2 m) was shifted to more positive potentials, and its voltage dependence reduced by 50% in the ∆K1500 mutant. To determine whether the positive charge deletion was the basis for the gating changes, we performed the mutations K1500Q and K1500E (∆ charge, -1, -2). For both mutations, V 1/2 h ∞ was shifted back toward control; however, V 1/2 m shifted progressively to more positive potentials. The late component of Na + current was increased in the ∆K1500 mutant channel. These changes can account for the complex phenotype in this kindred and point to an important role of the III/IV linker in channel activation.

The function of the 12 positive charges in the 53-residue III/IV interdomain linker of the cardiac Na(+) channel is unclear. We have identified a four-generation family, including 17 gene carriers with long QT syndrome, Brugada syndrome, and conduction system disease with deletion of lysine 1500 (DeltaK1500) within the linker. Three family members died suddenly. We have examined the functional consequences of this mutation by measuring whole-cell and single-channel currents in 293-EBNA cells expressing the wild-type and DeltaK1500 mutant channel. The mutation shifted V(1/2)h( infinity ) to more negative membrane potentials and increased k(h) consistent with a reduction of inactivation valence of 1. The shift in h( infinity ) was the result of an increase in closed-state inactivation rate (11-fold at -100 mV). V(1/2)m was shifted to more positive potentials, and k(m) was doubled in the DeltaK1500 mutant. To determine whether the positive charge deletion was the basis for the gating changes, we performed the mutations K1500Q and K1500E (change in charge, -1 and -2, respectively). For both mutations, V(1/2)h was shifted back toward control; however, V(1/2)m shifted progressively to more positive potentials. The late component of Na(+) current was increased in the DeltaK1500 mutant channel. These changes can account for the complex phenotype in this kindred and point to an important role of the III/IV linker in channel activation.

Abstract-Acute injury after adenoviral vascular gene transfer remains incompletely characterized. Here, we describe the early response (Յdays) in 52 New Zealand White rabbits undergoing gene transfer (␤-galactosidase or empty vector) or sham procedures to both carotid arteries. After gene transfer, arteries were either left in vivo for 1 hour to 3 days (in vivo arteries) or were excised immediately after gene transfer and cultured (ex vivo arteries). Within 1 hour, in vivo arteries receiving infectious titers of Ն4ϫ10 9 plaque-forming units (pfu)/mL showed endothelial activation, with an acute inflammatory infiltrate developing by 6 hours. Ex vivo arteries showed endothelial activation but no inflammatory infiltrate. There were also significant differences in transgene expression between in vivo and ex vivo arteries. Ex vivo arteries showed titer-dependent increases in ␤-galactosidase expression through 2ϫ10 10 pfu/mL, whereas in in vivo arteries, titers above 4ϫ10 9 pfu/mL merely increased acute inflammatory response, without increasing transgene expression. In vivo arteries showed significant time-and titer-dependent impairment in endothelium-dependent relaxation, with no effect on contraction or nitroprusside-induced relaxation. Interestingly, however, if rabbits were made neutropenic with vinblastine, their arteries maintained full endothelium-dependent relaxation, even after very high titer vascular infection (up to 1ϫ10 11 pfu/mL). These findings show that recombinant adenovirus triggers an early inflammatory response, and it is the inflammatory response that in turn causes functional endothelial injury. This occurs at much lower titers than previously appreciated (though the precise threshold will undoubtedly vary between laboratories). However, titers below the inflammatory threshold produce excellent transgene expression without inflammation or vascular injury. (Circ Res. 1998;82:1253-1262.)

Background-Hypercholesterolemia reduces nitric oxide bioavailability, manifested by reduced endothelium-dependent vascular relaxation, and also induces vascular adhesion molecule expression and inflammatory cell infiltration. We have previously shown that gene therapy with NO synthase in hypercholesterolemic rabbits substantially reverses the deficit in vascular relaxation. In the present study, we show that NO synthase gene therapy rapidly and substantially reduces vascular adhesion molecule expression, lipid deposition, and inflammatory cell infiltration. Methods and Results-Thirty male New Zealand White rabbits were maintained on a 1% cholesterol diet for 11 to 13 weeks, then underwent carotid artery gene transfer with Ad.nNOS or Ad.␤Gal (recombinant adenoviruses expressing neuronal NO synthase or ␤-galactosidase, respectively), or received medium alone in a sham procedure. Arteries were harvested at 1 and 3 days after gene transfer, and the following parameters were determined by immunohistochemical and image-analysis techniques: intercellular adhesion molecule-1, vascular cell adhesion molecule-1, lipid deposition by oil red O staining, lymphocyte infiltration (CD43-positive cells), and monocyte infiltration (RAM-11-positive cells). In Ad.nNOS-treated arteries, all markers were significantly decreased relative to Ad.␤Gal or sham-treated arteries within 3 days after gene transfer. Ad.nNOS had a particularly striking impact on monocyte infiltration; as early as 24 hours after gene transfer, Ad.nNOS-treated arteries had Ͼ3-fold fewer monocytes than Ad.␤Gal-or sham-treated arteries. Conclusions-NO synthase gene therapy rapidly ameliorates several markers of atherosclerosis in the cholesterol-fed rabbit. (Circulation. 1999;99:2979-2982.)

The function of the 12 positive charges in the 53-residue III/IV interdomain linker of the cardiac Na + channel is unclear. We have identified a four-generation family, including 17 gene carriers with long QT syndrome, Brugada syndrome, and conduction system disease with deletion of lysine 1500 (∆K1500) within the linker. Three family members died suddenly. We have examined the functional consequences of this mutation by measuring whole-cell and single-channel currents in 293-EBNA cells expressing the wild-type and ∆K1500 mutant channel. The mutation shifted the potential for half inactivation (V 1/2 h ∞ ) to more negative values and reduced its voltage dependence consistent with a reduction of inactivation valence of 1. The shift in inactivation was the result of an increase in closed-state inactivation rate (11-fold at -100 mV). The potential for half activation (V 1/2 m) was shifted to more positive potentials, and its voltage dependence reduced by 50% in the ∆K1500 mutant. To determine whether the positive charge deletion was the basis for the gating changes, we performed the mutations K1500Q and K1500E (∆ charge, -1, -2). For both mutations, V 1/2 h ∞ was shifted back toward control; however, V 1/2 m shifted progressively to more positive potentials. The late component of Na + current was increased in the ∆K1500 mutant channel. These changes can account for the complex phenotype in this kindred and point to an important role of the III/IV linker in channel activation.