Long QT syndrome (LQTS) is a heritable disease associated with ECG QT interval prolongation, ventricular tachycardia, and sudden cardiac death in young patients. Among genotyped individuals, mutations in genes encoding repolarizing K + channels (LQT1:KCNQ1; LQT2:KCNH2) are present in approximately 90% of affected individuals. Expression of pore mutants of the human genes KCNQ1 (KvLQT1-Y315S) and KCNH2 (HERG-G628S) in the rabbit heart produced transgenic rabbits with a long QT phenotype. Prolongations of QT intervals and action potential durations were due to the elimination of I Ks and I Kr currents in cardiomyocytes. LQT2 rabbits showed a high incidence of spontaneous sudden cardiac death (>50% at 1 year) due to polymorphic ventricular tachycardia. Optical mapping revealed increased spatial dispersion of repolarization underlying the arrhythmias. Both transgenes caused downregulation of the remaining complementary I Kr and I Ks without affecting the steady state levels of the native polypeptides. Thus, the elimination of 1 repolarizing current was associated with downregulation of the reciprocal repolarizing current rather than with the compensatory upregulation observed previously in LQTS mouse models. This suggests that mutant KvLQT1 and HERG interacted with the reciprocal wild-type α subunits of rabbit ERG and KvLQT1, respectively. These results have implications for understanding the nature and heterogeneity of cardiac arrhythmias and sudden cardiac death.

Testosterone regulates the expression of platelet TXA2 receptors in humans. This may contribute to the thrombogenicity of androgenic steroids.

Abstract-In this article we have investigated the mechanisms by which retrograde trafficking regulates the surface expression of the voltage-gated potassium channel, Kv1.5. Overexpression of p50/dynamitin, known to disrupt the dynein-dynactin complex responsible for carrying vesicle cargo, substantially increased outward K ϩ currents in HEK293 cells stably expressing Kv1.5 (0.57Ϯ0.07 nA/pF, nϭ12; to 1.18Ϯ0.2 nA/pF, nϭ12, PϽ0.01), as did treatment of the cells with a dynamin inhibitory peptide, which blocks endocytosis. Nocodazole pretreatment, which depolymerizes the microtubule cytoskeleton along which dynein tracks, also doubled Kv1.5 currents in HEK cells and sustained K ϩ currents in isolated rat atrial myocytes. These increased currents were blocked by 1 mmol/L 4-aminopyridine, and the specific Kv1.5 antagonist, DMM (100 nM). Confocal imaging of both HEK cells and myocytes, as well as experiments testing the sensitivity of the channel in living cells to external Proteinase K, showed that this increase of K ϩ current density was caused by a redistribution of channels toward the plasma membrane. Coimmunoprecipitation experiments demonstrated a direct interaction between Kv1.5 and the dynein motor complex in both heterologous cells and rat cardiac myocytes, supporting the role of this complex in Kv1.5 trafficking, which required an intact SH3-binding domain in the Kv1.5 N terminus to occur. These experiments highlight a pathway for Kv1.5 internalization from the cell surface involving early endosomes, followed by later trafficking by the dynein motor along microtubules. This work has significant implications for understanding the way Kv channel surface expression is regulated. (Circ Res. 2005;97:363-371.)Key Words: atrial myocyte Ⅲ cardiomyocytes Ⅲ intracellular protein transport Ⅲ ion channels Ⅲ potassium channels V oltage-gated K ϩ channels (Kv channels) are intimately involved in the cellular regulation of excitation in all cardiovascular cells, and their activity depends on the presence of active channel subunits at the plasmalemma. Surface expression is regulated by changes in gene expression, 1-3 interactions with accessory subunits, by phosphorylation, and by cellular components that regulate their trafficking to the cell surface. Trafficking can also provide an explanation for the mechanisms by which drugs may act to achieve their therapeutic actions. 4 Although several groups have investigated motifs within K ϩ channels that affect trafficking, [5][6][7][8] little is known about the molecules and machinery involved in these processes in the heart. Surface expression requires movement from the endoplasmic reticulum through the Golgi apparatus to the plasma membrane, and several studies have investigated channel determinants that affect this trafficking process. Motifs in the C termini and pore domains, 6,7 of Kv channels have been implicated in their differential surface expression presumably through effects on forward trafficking. 5 Functional expression of channels can also be regulated by the rem...

Using a PCR-based strategy and degenerate oligonucleotides, we isolated a Legionella pneumophila gene that showed high sequence similarity to members of the fliI gene family. An insertion mutation that disrupted the fliI open reading frame was recombined onto the L. pneumophila chromosome and analyzed for its effects on production of flagella and intracellular growth. The mutation resulted in loss of surface-localized flagellin protein but had no effect on the ability of the bacteria to grow within cultured cells. Therefore, in spite of the fact that some aflagellar mutations render L. pneumophila unable to grow within macrophages, the isolation of this defined mutant confirms that production of flagella is not required for intracellular growth.

Testosterone has been implicated as a risk factor for cardiovascular diseases. Thromboxane (Tx) A2 is an important pathophysiological mediator for thrombotic vascular diseases. This study investigated the effects of testosterone on platelet and vascular TxA2 receptors. Male rats were treated with either testosterone cypionate for 2 wk, sham operated, castrated, or castrated and treated with testosterone cypionate for 2 wk. Treatment of intact rats with testosterone significantly (P < 0.001) increased the TxA2 receptor density in platelets from 25.4 +/- 3.2 to 42.9 +/- 4.2 fmol/mg protein (P < 0.005, n = 17) and in aortic membranes from 48.7 +/- 1.7 to 86.1 +/- 6.1 fmol/mg protein, n = 9. The threshold concentration of the TxA2 mimetic, [1S-(1 alpha, 2 beta(5Z),3 alpha(1E,3R*)4 alpha)]-7-[3-(3-hydroxy-4- (4'-iodophenoxy)-1-butenyl)-7-oxabicyclo[2.21]heptan-2-yl]-5 -heptenoic acid (I-BOP), to induce platelet aggregation was significantly (P < 0.01) decreased from 0.45 +/- 0.16 nM, n = 7, in the control rats to 0.07 +/- 0.01 nM, n = 13, in the testosterone-treated rats. Testosterone treatment resulted in a significantly (P < 0.05) greater maximum aortic contractile response to the TxA2 mimetic, U-46619, compared with intact rats. Castration resulted in a significant (P < 0.01) decrease in aortic TxA2 receptor density from 51.7 +/- 3.7 to 27.3 +/- 5.3 fmol/mg protein, which was significantly reversed by testosterone treatment (89.2 +/- 7.1 fmol/mg protein; n = 4). Castration resulted in a significantly (P < 0.05) lower maximal aortic contractile response that was reversed by treatment with testosterone. Castration did not significantly change platelet TxA2 receptor density.(ABSTRACT TRUNCATED AT 250 WORDS)

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Structural models of voltage-gated channels suggest that flexibility of the S3-S4 linker region may be important in allowing the S4 region to undergo large conformational changes in its putative voltage-sensing function. We report here the initial characterization of 18 mutations in the S3-S4 linker of the Shaker channel, including deletions, insertions, charge changes, substitution of prolines, and chimeras replacing the 25-residue Shaker linker with 7-or 9-residue sequences from Shab , Shaw , or Shal . As measured in Xenopus oocytes with a two-microelectrode voltage clamp, each mutant construct yielded robust currents. Changes in the voltage dependence of activation were small, with activation voltage shifts of 13 mV or less. Substitution of linkers from the slowly activating Shab and Shaw channels resulted in a three-to fourfold slowing of activation and deactivation. It is concluded that the S3-S4 linker is unlikely to participate in a large conformational change during channel activation. The linker, which in some channel subfamilies has highly conserved sequences, may however be a determinant of activation kinetics in potassium channels, as previously has been suggested in the case of calcium channels. The high sensitivity of voltage-gated potassium and sodium channels arises from a large charge displacement, ‫ف‬ 13 e 0 in magnitude, as a channel moves from its resting state to the open state (Schoppa et al., 1992;Hirschberg et al., 1995;Aggarwal and Mackinnon, 1996;Seoh et al., 1996). It is widely thought that this charge displacement involves the S4 region of the protein sequence, with contributions from other regions (Sigworth, 1994;Seoh et al., 1996). The S4 region of the Shaker potassium channel has seven basic residues which could contribute to the mobile charge, and recent experiments have shown changes in the accessibility of S4 residues upon voltage-dependent channel activation (Larsson et al., 1996;Yang et al., 1996). According to some theories, the S4 segment is an alpha helix that undergoes a large, helical screw movement normal to the lipid bilayer in order to transfer the necessary gating charge (Catterall, 1986;Durell et al. 1992). If indeed this is how the S4 region moves, then the S3-S4 linker, to which it is tethered at its NH 2 -terminal end, must be flexible to support the large displacement.There has been little study of the S3-S4 linker of voltage-gated channels, apart from work on L-type calcium channels by Nakai et al. (1994). These authors showed that exchange of the S3-S4 region in Domain 1 changed the channel from the skeletal muscle (slowly activating) to the cardiac (rapidly activating) kinetic behavior. A comparison of potassium channel S3-S4 linkers is shown in Fig. 1. The apparent length of the linker is variable between subfamilies, ranging from 25 residues for Drosophila Shaker to only 7 residues in Shaw . The members of the Shaker subfamily show a moderate degree of sequence identity, but within the Shab and Shal subfamilies there is a remarkable degree of identi...