Proline scan of the HERG channel S6 helix reveals the location of the intracellular pore gate.

In Shaker-like channels, the activation gate is formed at the bundle crossing by the convergence of the inner S6 helices near a conserved proline-valine-proline motif, which introduces a kink that allows for electromechanical coupling with voltage sensor motions via the S4-S5 linker. Human ether-a-go-go-related gene (hERG) channels lack the proline-valine-proline motif and the location of the intracellular pore gate and how it is coupled to S4 movement is less clear. Here, we show that proline substitutions within the S6 of hERG perturbed pore gate closure, trapping channels in the open state. Performing a proline scan of the inner S6 helix, from Ile(655) to Tyr(667) revealed that gate perturbation occurred with proximal (I655P-Q664P), but not distal (R665P-Y667P) substitutions, suggesting that Gln(664) marks the position of the intracellular gate in hERG channels. Using voltage-clamp fluorimetry and gating current analysis, we demonstrate that proline substitutions trap the activation gate open by disrupting the coupling between the voltage-sensing unit and the pore of the channel. We characterize voltage sensor movement in one such trapped-open mutant channel and demonstrate the kinetics of what we interpret to be intrinsic hERG voltage sensor movement.

Virtual monoenergetic reconstruction of contrast-enhanced CT scans of the abdomen and pelvis at 40 keV improves the detection of peritoneal metastatic deposits.

PURPOSE: To evaluate the role of virtual monoenergetic imaging (VMI) in the detection of peritoneal metastatic disease in contrast-enhanced computed tomography (CT) of the abdomen and pelvis and to compare this technique to the conventional 120 kV mixed dataset. MATERIALS AND METHODS: Institutional review board approval was obtained with no informed consent required for this retrospective analysis. 43 consecutive patients with histopathologically confirmed peritoneal disease were scanned using a standard protocol on a 128-section dual-source, dual-energy CT system (100/140 keV). Scans were retrospectively reconstructed at VMI energy levels from 40-110 keV in 10 keV increments and were analyzed both quantitatively and qualitatively. CNR values for peritoneal metastatic deposits were recorded using region of interest (ROI) analysis at each energy level for all VMI datasets. Subjective analysis was performed by two independent fellowship-trained readers with combined experience of greater than 15 years. Qualitative parameters included diagnostic acceptability, subjective noise, and contrast resolution and confidence. RESULTS: The contrast-to-noise ratios (CNRs) for peritoneal metastatic deposits at the different VMI energy levels were compared using a one-way ANOVA with Tukey Post Test, and the optimal CNR was observed at 40 keV (p < 0.0001). Qualitative parameters were compared using a Paired T Test. Subjective noise, diagnostic acceptability, and contrast resolution was significantly better on the conventional images, but readers reported increased confidence on VMI at 40 keV (p < 0.001). CONCLUSION: VMI reconstruction of contrast-enhanced dual-energy CT scans of the abdomen and pelvis at 40 keV maximizes the conspicuity of metastatic peritoneal deposits and improves radiologists' diagnostic confidence compared with conventional CT images. We recommend using virtual monoenergetic datasets at 40 keV as a tool for improving the detection of these lesions in routine clinical practice.