Structural and functional analysis of critical amino acids in TMVI of the NHE1 isoform of the Na+/H+ exchanger

AbstractThe mammalian Na+/H+ exchanger isoform 1 (NHE1) resides on the plasma membrane and exchanges one intracellular H+ for one extracellular Na+. It maintains intracellular pH and regulates cell volume, and cell functions including growth and cell differentiation. Previous structural and functional studies on TMVI revealed several amino acids that are potentially pore lining. We examined these and other critical residues by site-directed mutagenesis substituting Asn227→Ala, Asp, Arg; Ile233→Ala; Leu243→Ala; Glu247→Asp, Gln; Glu248→Asp, Gln. Mutant NHE1 proteins were characterized in AP-1 cells, which do not express endogenous NHE1. All the TMVI critical amino acids were highly sensitive to substitution and changes often lead to a dysfunctional protein. Mutations of Asn227→Ala, Asp, Arg; Ile233→Ala; Leu243→Ala; Glu247→Asp; Glu248→Gln yielded significant reduction in NHE1 activity. Mutants of Asn227 demonstrated defects in protein expression, targeting and activity. Substituting Asn227→Arg and Ile233→Ala decreased the surface localization and expression of NHE1 respectively. The pore lining amino acids Ile233 and Leu243 were both essential for activity. Glu247 was not essential, but the size of the residue at this location was important while the charge on residue Glu248 was more critical to NHE1 function. Limited trypsin digestion on Leu243→Ala and Glu248→Gln revealed that they had increased susceptibility to proteolytic attack, indicating an alteration in protein conformation. Modeling of TMVI with TMXI suggests that these TM segments form part of the critical fold of NHE1 with Ile233 and Leu465 of TMXI forming a critical part of the extracellular facing ion conductance pathway

Adaptive Changes of the Ulnar Collateral Ligament of Professional Baseball from Different Climates Over Multiple Seasons: An 18-Year Study

Background: The ulnar collateral ligament (UCL) is put under high levels of stress through the overhead throwing motion typical of professional baseball pitchers. In response to repetitive stress the UCL undergoes adaptive changes including thickening of the ligament and increased joint laxity under valgus stress. Studies examining the adaptive changes in the elbows of professional pitchers over multiple seasons are limited. Investigations such as this one offer insight into the changes in the UCL of professional pitchers over multiple years of professional play as well as the risk for injuries that may be associated with these changes. Purpose/Hypothesis: The purpose of this study was to compare stress ultrasound findings between warm and cold climate pitchers and to assess for significant differences that may predispose players from either climate to future injury. Study Design: Retrospective Cohort Study Methods: Dynamic stress ultrasounds (SUS) of professional baseball pitchers were taken over an 18 year period. Player demographics were collected to determine climate of origin. SUS measurements of the same player taken 3 years apart were examined to determine changes in relative UCL thickness and laxity between the dominant and nondominant arms. The term ‘relative’ refers to (dominant – non-dominant), laxity (joint space distance under stress - joint space distance at rest). SUS were also examined at time of measurements for pathologic findings. Results: Players from colder and warmer climates did not differ from one another in the progression of relative UCL thickness (0.72 mm vs 0.55 mm, P = .748) of relative laxity (-0.01 mm vs 0.03 mm, P = .904). Both groups did not differ in rates of pathology development including calcifications (P = .412), hypoechoic foci (P = .084), osteophyte (P = .892). Conclusion: Adaptive changes of the UCL in professional baseball pitchers originating from colder and warmer climates did not significantly differ from one another. Although there is potential for those from warmer climates to throw year-round the effect may not be enough to cause noticeable changes over a 3 year period. Future studies should expand on this investigation with examination of injuries of players from different climates to determine if any correlation exists with UCL changes in an expanded cohort

Impact of Draft Order on Stress Sonography of the Ulnar Collateral Ligament of the Elbow in Professional Baseball Pitchers: An 18-Year Study

Background: The MLB draft is a common route for players to enter professional baseball in the United States. Individuals taken in earlier rounds are typically higher-performing players. When looking at pitchers specifically, higher performance at the amateur level may be associated with increased frequency of adaptive change in the throwing elbow. Purpose/Hypothesis: To determine if players taken in earlier rounds of the MLB draft have a greater frequency or extent of pathological change in the elbow, as measured by dynamic stress ultrasound. Study Design: Retrospective Cohort Study Methods: Dynamic stress ultrasounds (SUS) were performed over an 18-year period on the dominant and nondominant arms of 651 professional pitchers. The 383 players who were drafted were grouped according to which round they were drafted in (rounds 1-5, 6-10, 11-20, 21+). Groups were compared with respect to “relative” ulnar collateral ligament thickness (dominant – nondominant), “relative” ulnohumeral joint laxity (joint space distance under stress – distance at rest), and the presence of pathology (calcifications, tears, hypoechoic foci, osteophytes). Additionally, a subgroup analysis was done to compare progression of SUS findings over a 3-year period in players for which data was available. Results: Draft round groups did not differ by age, number of prior spring trainings, or handedness. Comparing baseline measurements, there was no significant relationship between draft round and relative UCL thickness (P = .932), relative laxity (P = .996), or presence of pathology detectable on SUS (P = .642). However, increased relative UCL thickness was significantly associated with the presence of pathology on SUS (OR: 1.45, 95% CI [1.26-1.69], P \u3c .001). Longitudinally, there was no significant relationship between draft round and 3-year progression of relative laxity, relative UCL thickness, or clinical progression of pathology. Conclusion: Higher-performing pitchers are drafted earlier in the MLB draft. This may be attributable to peak pitch velocity, in-game performance, visibility gained during player showcases, or any number of other sport-specific variables. However, despite this, there was no significant relationship between draft round and adaptive changes to the elbow on SUS

Structural and functional analysis of extracellular loop 4 of the Nhe1 isoform of the Na+/H+ exchanger

AbstractThe mammalian Na+/H+ exchanger isoform 1 (NHE1) is a ubiquitously expressed plasma membrane protein. It regulates intracellular pH by removing a single intracellular H+ in exchange for one extracellular Na+. The membrane domain of NHE1 comprises the 500 N-terminal amino acids and is made of 12 transmembrane segments. The extracellular loops of the transmembrane segments are thought to be involved in cation coordination and inhibitor sensitivity. We have characterized the structure and function of amino acids 278–291 representing extracellular loop 4. When mutated to Cys, residues F277, F280, N282 and E284 of EL4 were sensitive to mutation and reaction with MTSET inhibiting NHE1 activity. In addition they were found to be accessible to extracellular applied MTSET. A peptide of the amino acids of EL4 was mostly unstructured suggesting that it does not provide a rigid structured link between TM VII and TM VIII. Our results suggest that EL4 makes an extension upward from TM VII to make up part of the mouth of the NHE1 protein and is involved in cation selectivity or coordination. EL4 provides a flexible link to TM VIII which may either allow movement of TM VII or allow TM VIII to not be adjacent to TM VII

Structural and Functional Analysis of the Transmembrane Segment Pair VI and VII of the NHE1 Isoform of the Na⁺/H⁺ Exchanger

Isoform 1 of the mammalian Na⁺/H⁺ exchanger (NHE1) is a ubiquitously expressed plasma membrane pH regulatory protein. It removes one intracellular H⁺ in exchange for one extracellular Na⁺. The 500 N-terminal amino acids comprise the catalytic membrane domain and fold into 12 transmembrane (TM) segments. To gain insight into the structure and function of human NHE1, a region spanning transmembrane domains VI and VII was expressed and purified, and the structure was determined using nuclear magnetic resonance (NMR). Segment VI includes two structurally conserved regions corresponding to two short α-helices involving residues 229–236 and 239–247. Segment VII includes one long helical region spanning residues 255–274. The NMR structure of the peptide containing transmembrane domains VI and VII was very similar to the previously published structures of the single-transmembrane segments except that TM VII was not kinked. Tryptophan scanning site-directed mutagenesis of TM VI demonstrated that mutation of residues V240–V245 to tryptophan eliminated NHE1 activity when the full length protein was expressed in cells. In contrast, mutants F246W and E247W were functional. Double mutant V242F/F260V retained activity, while the individual mutations were not active. The results suggest that the region of TM VI from V240 to V245 is closely associated with TM VII and that, in agreement with the NMR structure of VI–VII segments, V242 and F260 are in close association. A study of two transmembrane peptides provides further insight into the structure of the NHE1 protein

An Isolated Midsubstance ACL Tear Repaired With the BEAR System

Background: Anterior cruciate ligament (ACL) repairs, once widely abandoned due to historically high failure rates, have recently regained interest with the development of the bridge-enhanced ACL repair (BEAR) implant, a novel arthroscopic technique that uses a resorbable protein-based implant combined with autologous blood to primarily repair a midsubstance ACL tear. This technical note presents a step-by-step surgical method for performing an isolated midsubstance ACL repair using the BEAR implant. Indications: The BEAR implant is indicated for skeletally mature patients at least 14 years of age with a complete rupture of the ACL, confirmed by magnetic resonance imaging. The complete ACL tear must have an attached stump to the tibia. Technique Description: A diagnostic arthroscopy is used to confirm complete rupture of the ACL and presence of residual tibial stump. A self-retrieving suture passage device is used to whipstitch a total of 6 passes with #2 Vicryl suture from distal to proximal through the residual stump. A notchplasty followed by femoral and tibial tunnels is created in a standard fashion. An Endobutton, soaked in a bacitracin solution, is then loaded with the sutures that were previously passed through the residual ACL stump and then through the femoral tunnel and cinched down to bone. The 4 suture ends that are coming from the Endobutton are then passed through the BEAR implant with the use of a Keith needle and shuttled through the tibial tunnel. The BEAR implant is hydrated with 15 cc of the patient's blood and is shuttled through the anteromedial portal with the knee in full extension. The 4 tibial sutures passed through the graft and tibial tunnel are passed, and tensioned to the proximal tibial with a second Endobutton. Standard closure and dressings are applied. Results: This new surgical implant and technique have shown noninferiority to ACL autograft reconstruction with respect to the International Knee Documentation Committee and anteroposterior laxity, with improved hamstring strength and decreased incidence of contralateral ACL tears at 2 years postoperative. Discussion/Conclusion: While initial data remain promising, future long-term designed studies are needed to determine the clinical efficacy of the BEAR technique, particularly comparing itself with bone-patellar tendon-bone autograft ACL reconstruction. Patient Consent Disclosure Statement: The author(s) attests that consent has been obtained from any patient(s) appearing in this publication. If the individual may be identifiable, the author(s) has included a statement of release or other written form of approval from the patient(s) with this submission for publication

Structural and Functional Analysis of Transmembrane Segment VI of the NHE1 Isoform of the Na+/H+ Exchanger*

The Na+/H+ exchanger isoform 1 is a ubiquitously expressed integral membrane protein. It resides on the plasma membrane of cells and regulates intracellular pH in mammals by extruding an intracellular H+ in exchange for one extracellular Na+. We characterized structural and functional aspects of the transmembrane segment (TM) VI (residues 227–249) by using cysteine scanning mutagenesis and high resolution NMR. Each residue of TM VI was mutated to cysteine in the background of the cysteineless NHE1 protein, and the sensitivity to water-soluble sulfhydryl-reactive compounds (2-(trimethylammonium)ethyl)methanethiosulfonate (MTSET) and (2-sulfonatoethyl)methanethiosulfonate (MTSES) was determined for those residues with significant activity remaining. Three residues were essentially inactive when mutated to Cys: Asp238, Pro239, and Glu247. Of the remaining residues, proteins with the mutations N227C, I233C, and L243C were strongly inhibited by MTSET, whereas amino acids Phe230, Gly231, Ala236, Val237, Ala244, Val245, and Glu248 were partially inhibited by MTSET. MTSES did not affect the activity of the mutant NHE1 proteins. The structure of a peptide representing TM VI was determined using high resolution NMR spectroscopy in dodecylphosphocholine micelles. TM VI contains two helical regions oriented at an approximate right angle to each other (residues 229–236 and 239–250) surrounding a central unwound region. This structure bears a resemblance to TM IV of the Escherichia coli protein NhaA. The results demonstrate that TM VI of NHE1 is a discontinuous pore-lining helix with residues Asn227, Ile233, and Leu243 lining the translocation pore