Nathan Wong

Kif5b is an essential forward trafficking motor for the Kv1.5 cardiac potassium channel

We have investigated the role of the kinesin I isoform Kif5b in the trafficking of a cardiac voltage‐gated potassium channel, Kv1.5. In Kv1.5‐expressing HEK293 cells and H9c2 cardiomyoblasts, current densities were increased from control levels of 389 ± 50.0 and 317 ± 50.3 pA pF−1, respectively, to 614 ± 74.3 and 580 ± 90.9 pA pF−1 in cells overexpressing the Kif5b motor. Overexpression of the Kif5b motor increased Kv1.5 expression additively with several manipulations that reduce channel internalization, suggesting that it is involved in the delivery of the channel to the cell surface. In contrast, expression of a Kif5b dominant negative (Kif5bDN) construct increased Kv1.5 expression non‐additively with these manipulations. Thus, the dominant negative acts by indirectly inhibiting endocytosis. The increase in Kv1.5 currents induced by wild‐type Kif5b was dependent on Golgi function; a 6 h treatment with Brefeldin A reduced Kv1.5 currents to control levels in Kif5b‐overexpressing cells but had little effect on the increase associated with Kif5bDN expression. Finally, expression of the Kif5bDN prior to induction of Kv1.5 in a tetracycline inducible system blocked surface expression of the channel in both HEK293 cells and H9c2 cardiomyoblasts. Thus, Kif5b is essential to anterograde trafficking of a cardiac voltage‐gated potassium channel.

Microscopic mechanisms for long QT syndrome type 1 revealed by single-channel analysis of IKs with S3 domain mutations in KCNQ1

BACKGROUND The slowly activating delayed rectifier current IKs participates in cardiac repolarization, particularly at high heart rates, and mutations in this K(+) channel complex underlie long QT syndrome (LQTS) types 1 and 5. OBJECTIVE The purpose of this study was to determine biophysical mechanisms of LQT1 through single-channel kinetic analysis of IKs carrying LQT1 mutations in the S3 transmembrane region of the pore-forming subunit KCNQ1. METHODS We analyzed cell-attached recordings from mammalian cells in which a single active KCNQ1 (wild type or mutant) and KCNE1 complex could be detected. RESULTS The S3 mutants of KCNQ1 studied (D202H, I204F, V205M, and S209F), with the exception of S209F, all led to a reduction in channel activity through distinct kinetic mechanisms. D202H, I204F, and V205M showed decreased open probability (Po) compared with wild type (0.07, 0.04, and 0.12 vs 0.2); increased first latency from 1.66 to >2 seconds at +60 mV (I204F, V205M); variable-to-severe reductions in open dwell times (≥50% in V205M); stabilization of closed states (D202H); and an inability of channels to reach full conductance levels (V205M, I204F). S209F is a kinetic gain-of-function mutation with a high Po (0.40) and long open-state dwell times. CONCLUSION S3 mutations in KCNQ1 cause diverse kinetic defects in I(Ks), affecting opening and closing properties, and can account for LQT1 phenotypes.

Control of voltage-gated K+ channel permeability to NMDG+ by a residue at the outer pore

Crystal structures of potassium (K+) channels reveal that the selectivity filter, the narrow portion of the pore, is only ∼3-Å wide and buttressed from behind, so that its ability to expand is highly constrained, and the permeation of molecules larger than Rb+ (2.96 Å in diameter) is prevented. N-methyl-d-glucamine (NMDG+), an organic monovalent cation, is thought to be a blocker of Kv channels, as it is much larger (∼7.3 Å in mean diameter) than K+ (2.66 Å in diameter). However, in the absence of K+, significant NMDG+ currents could be recorded from human embryonic kidney cells expressing Kv3.1 or Kv3.2b channels and Kv1.5 R487Y/V, but not wild-type channels. Inward currents were much larger than outward currents due to the presence of intracellular Mg2+ (1 mM), which blocked the outward NMDG+ current, resulting in a strong inward rectification. The NMDG+ current was inhibited by extracellular 4-aminopyridine (5 mM) or tetraethylammonium (10 mM), and largely eliminated in Kv3.2b by an S6 mutation that prevents the channel from opening (P468W) and by a pore helix mutation in Kv1.5 R487Y (W472F) that inactivates the channel at rest. These data indicate that NMDG+ passes through the open ion-conducting pore and suggest a very flexible nature of the selectivity filter itself. 0.3 or 1 mM K+ added to the external NMDG+ solution positively shifted the reversal potential by ∼16 or 31 mV, respectively, giving a permeability ratio for K+ over NMDG+ (PK+/PNMDG+) of ∼240. Reversal potential shifts in mixtures of K+ and NMDG+ are in accordance with PK+/PNMDG+, indicating that the ions compete for permeation and suggesting that NMDG+ passes through the open state. Comparison of the outer pore regions of Kv3 and Kv1.5 channels identified an Arg residue in Kv1.5 that is replaced by a Tyr in Kv3 channels. Substituting R with Y or V allowed Kv1.5 channels to conduct NMDG+, suggesting a regulation by this outer pore residue of Kv channel flexibility and, as a result, permeability.

Nasofrontal outflow tract visibility in computed tomography imaging of frontal sinus fractures.

The choice of frontal sinus fracture treatment is based on multiple factors, one of which is injury to the nasofrontal outflow tract (NFOT). Computed tomography (CT) imaging of the NFOT can play an important role in the decision process. We sought to assess the visibility of the NFOT on CT scans in frontal sinus fractures. Patients with frontal sinus fractures (including the posterior table) receiving a CT scan from April 1st 2001 to December 31st 2009 were included. Scans were retrospectively assessed for available views (axial, coronal, and sagittal), slice thickness, inclusion of the anatomical NFOT region in the scanned area, and visibility of the NFOT. A total of 170 patients were included. In majority (71%) of patients NFOT was visible on one or more views, whereas in 33% (N = 56) of patients had three complete views (complete anatomical NFOT region scanned in three views). In this subgroup, the ability to assess the NFOT increased to 89%. When selecting patients with three complete views of ≤ 2 mm slice thickness (N = 47), the ability to assess the NFOT increased to 96%. In conclusion, when assessing the NFOT using CT imaging, having three complete views (axial, coronal, and sagittal) and a ≤ 2 mm slice thickness greatly increases the NFOT visibility.

Continuous antibiotic prophylaxis in the setting of prenatal hydronephrosis and vesicoureteral reflux

Continuous antibiotic prophylaxis (CAP) has traditionally been offered for children with recurrent urinary tract infections (UTIs) or those at risk, including children diagnosed with prenatal hydronephrosis (HN) and vesicoureteral reflux (VUR). However, indications for antibiotic prophylaxis are controversial, data on who should benefit from this therapy is conflicting and, thus, guidelines are unable to provide conclusive recommendations. In the setting of prenatal HN, although randomized trials are currently underway, most evidence is derived from low- to moderate-quality observational studies. Although there is no benefit in those with low-grade HN, a systematic review of the available studies showed that high-grade HN patients on prophylaxis experienced fewer infections with an estimated number needed to treat of 7. On the other hand, there are eight randomized trials that have investigated the use of antibiotic prophylaxis in the setting of VUR. Although four of the studies have demonstrated some value of prophylaxis and the other four have not, meta-analysis has shown an overall benefit of antibiotic prophylaxis in preventing infections. The observed differences are likely due to different inclusion criteria and study heterogeneity. Although generalizing results of meta-analyses to all children is tempting, an individualized approach, by determining which patients best behave like those of the included studies, is recommended.

Genitourinary tuberculosis masquerading as a ureteral calculus.

The genitourinary tract is a common extrapulmonary site of tuberculosis infection, yet remains a rare clinical entity in North America. We report the case of a 37-year-old man who presented for extracorporeal shock wave lithotripsy for a suspected ureteral stone on imaging. Further workup confirmed a diagnosis of genitourinary tuberculosis. Medical management was undertaken and, ultimately, nephrectomy performed. This case highlights the importance of maintaining a high index of clinical suspicion for genitourinary tuberculosis.