Potassium channels are responsible for the selective permeation of K+ ions across cell membranes. K+ ions permeate in single file through the selectivity filter, a narrow pore lined by backbone carbonyls that compose 4 K+ binding sites. Here, we report 2D IR spectra of a semisynthetic KcsA channel with site-specific 13C18O isotope labels in the selectivity filter. The ultrafast time-resolution of 2D IR spectroscopy provides an instantaneous snapshot of the multi-ion configurations and structural distributions that occur spontaneously in the filter. Two elongated features are resolved, revealing the statistical weighting of two structural conformations. The spectra are reproduced by MD simulations of structures with water separating two K+ ions in the binding sites, ruling out configurations with ions occupying adjacent sites.

The selectivity filter of K+ channels contains four ion binding sites (S1–S4) and serves dual functions of discriminating K+ from Na+ and of acting as a gate during C-type inactivation. C-type inactivation is modulated by ion binding to the selectivity filter sites but the underlying mechanism is not known. Here we evaluate how the ion binding sites in the selectivity filter of the KcsA channel participate in C-type inactivation and in recovery from inactivation. We use unnatural amide-to-ester substitutions in the protein backbone to manipulate the S1–S3 sites and a side chain substitution to perturb the S4 site. We develop an improved semisynthetic approach for generating these amide-to-ester substitutions in the selectivity filter. Our combined electrophysiological and X-ray crystallographic analysis of the selectivity filter mutants show that the ion binding sites play specific roles during inactivation and provide insights into the structural changes at the selectivity filter during C-type inactivation.

The interplay between the intracellular gate and the selectivity filter underlies the structural basis for gating in potassium ion channels. Using a combination of protein semisynthesis, two-dimensional infrared (2D IR) spectroscopy, and molecular dynamics (MD) simulations, we probe the ion occupancy at the S1 binding site in the constricted state of the selectivity filter of the KcsA channel when the intracellular gate is open and closed. The 2D IR spectra resolve two features, whose relative intensities depend on the state of the intracellular gate. By matching the experiment to calculated 2D IR spectra of structures predicted by MD simulations, we identify the two features as corresponding to states with S1 occupied or unoccupied by K+. We learn that S1 is >70% occupied when the intracellular gate is closed and <15% occupied when the gate is open. Comparison of MD trajectories show that opening of the intracellular gate causes a structural change in the selectivity filter, which leads to a change in the ion occupancy. This work reveals the complexity of the conformational landscape of the K+ channel selectivity filter and its dependence on the state of the intracellular gate.

Objectives/Hypothesis The time interval at which Nasal Obstruction Symptom Evaluation (NOSE) scores stabilize after functional septorhinoplasty has not been determined. Our goal was to characterize longitudinal trends of patient‐reported outcomes of nasal obstruction using the NOSE survey instrument following functional septorhinoplasty. Study Design Prospective longitudinal cohort study. Methods Adult patients (≥18 years) with nasal obstruction who underwent functional septorhinoplasty by three different surgeons at a single academic, tertiary referral center were identified. NOSE scores were obtained preoperatively and prospectively during three postoperative intervals defined as early (1–3 months), middle (4–6 months), and late (≥10 months.) Longitudinal analysis included repeated measures analysis of variance and adjustments for multiple comparisons. Results A total of 49 patients met inclusion criteria. For the total cohort, mean NOSE scores significantly improved between preoperative and early postoperative evaluations (71.4, standard deviation [SD] ± 17.0 vs. 24.2, SD ± 19.5; P < .001) but did not significantly change between early and middle (20.6, SD ± 19.1; P = .543) or middle and late (23.1, SD ± 24.9; P > .999) time intervals. Conclusions Patients with nasal obstruction who undergo functional septorhinoplasty can be expected to have significant improvement in self ‐reported nasal obstruction as early as 1 to 3 months postoperatively with a continued, durable, long‐standing benefit lasting at least 10 months after surgery. Future studies can consider the 3‐month time frame as a proxy for 1 year outcomes to help reduce survey burden. Level of Evidence 2c Laryngoscope, 129:841–846, 2019

Background: Phalloplasty often requires free tissue transfer. There is ample literature describing flap-related outcomes, but the microsurgical technique used, including choice of recipient vessels, has been an overlooked yet important topic. In this study, the authors review the outcomes of their experience with the deep inferior epigastric artery and locoregional veins and outline technical modifications that occurred during the study period. Methods: A retrospective chart analysis of patients who underwent microsurgical phalloplasty between September of 2016 and July of 2019 was performed. Variables included flap design, donor site, and recipient vessels. The outcome measures were return to the operating room for flap compromise and partial or complete flap loss. Results: Forty-two phalloplasties using the deep inferior epigastric artery were identified. There were six take-backs for flap compromise, and four patients required venous revision, one of whom lost his urethral flap on postoperative day 9. There was a decrease in take-back rate from 30 percent in the first 20 patients to 0 percent in the second 22 patients in the study period. A total of 11.9 percent of patients had partial flap loss. This decreased from 15 percent to 9 percent in the two groups. Conclusion: After an initial learning curve, the combination of deep inferior epigastric artery, deep inferior epigastric vein, and great saphenous vein combined with specific technical modifications such as targeted coagulation of the vasa nervorum of the clitoral nerve has proven to be a reliable technique. CLINICAL QUESTION/LEVEL OF EVIDENCE: Therapeutic, IV.

Glutamate transporters catalyze the concentrative uptake of glutamate from synapses and are essential for normal synaptic function. Despite extensive investigations of glutamate transporters, the mechanisms underlying substrate recognition, ion selectivity and the coupling of substrate and ion transport are not well understood. Deciphering these mechanisms requires the ability to precisely engineer the transporter. In this study, we describe the semisynthesis of GltPh, an archaeal homolog of glutamate transporters. Semisynthesis enables the precise engineering of GltPh through the incorporation of unnatural amino acids and peptide backbone modifications. In the semisynthesis, the GltPh polypeptide is initially assembled from a recombinantly expressed thioester peptide and a chemically synthesized peptide using the native chemical ligation reaction followed by in vitro folding to the native state. We have developed a robust procedure for the in vitro folding of GltPh. Biochemical characterization of the semisynthetic GltPh indicates that it is similar to the native transporter. We used semisynthesis to substitute Arg397, a highly conserved residue in the substrate binding site with the unnatural analog, citrulline. Our studies demonstrate that Arg397 is required for high affinity substrate binding and based on our results we propose that Arg397 is involved in a Na+- dependent remodeling of the substrate binding site required for high affinity Asp binding. We anticipate that the semisynthetic approach developed in this study will be extremely useful in investigating functional mechanisms in GltPh. Further, the approach developed in this study should also be applicable to other membrane transport proteins.

Chest surgery for non-binary patients comprises a significant proportion of our transgender surgery practice, and surgeons providing affirming care should be familiar with the unique characteristics and treatment options for this patient population.

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