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The spectrin membrane skeleton controls the disposition of selected membrane channels, receptors, and transporters. In the brain [beta]III spectrin binds directly to the excitatory amino acid transporter (EAAT4), the glutamate receptor delta, and other proteins. Mutations in [beta]III spectrin link strongly to human spinocerebellar ataxia type 5 (SCA5), correlating with alterations in EAAT4. We have explored the mechanistic basis of this phenotype by targeted gene disruption of Spnb3. Mice lacking intact [beta]III spectrin develop normally. By 6 months they display a mild nonprogressive ataxia. By 1 year most [Spnb3.sup.-/-] animals develop a myoclonic seizure disorder with significant reductions of EAAT4, EAAT1, GluR[delta], IP3R, and NCAM140. Other synaptic proteins are normal. The cerebellum displays increased dark Purkinje cells (PC), a thin molecular layer, fewer synapses, a loss of dendritic spines, and a 2-fold expansion of the PC dendrite diameter. Membrane and expanded Golgi profiles fill the PC dendrite and soma, and both regions accumulate EAAT4. Correlating with the seizure disorder are enhanced hippocampal levels of neuropeptide Y and EAAT3 and increased calpain proteolysis of [alpha]II spectrin. It appears that [beta]III spectrin disruption impairs synaptogenesis by disturbing the intracellular pathways selectively regulating protein trafficking to the synapse. The mislocalization of these proteins secondarily disrupts glutamate transport dynamics, leading to seizures, neuronal damage, and compensatory changes in EAAT3 and neuropeptide Y. cytoskeleton | membrane | spinocerebellar ataxia type 5 | excitatory amino acid transporter 4 | Purkinje doi/10.1073/pnas.1001522107

Author(s): Adriana Donovan [1]; Alison Brownlie [1, 2]; Yi Zhou [1]; Jennifer Shepard [1]; Stephen J. Pratt [3]; John Moynihan [1]; Barry H. Paw [1]; Anna Drejer [1]; Bruce Barut [...]

Our goal was to identify aspects of residency applications predictive of subsequent performance during pediatric internship. We conducted a retrospective cohort study of graduates of US medical schools who began pediatric internship in a large pediatric residency program in the summers of 2013 to 2017. The primary outcome was the weighted average of subjects' Accreditation Council for Graduate Medical Education pediatric Milestone scores at the end of pediatric internship. To determine factors independently associated with performance, we conducted multivariate linear mixed-effects models controlling for match year and Milestone grading committee as random effects and the following application factors as fixed effects: letter of recommendation strength, clerkship grades, medical school reputation, master's or PhD degrees, gender, US Medical Licensing Examination Step 1 score, Alpha Omega Alpha membership, private medical school, and interview score. Our study population included 195 interns. In multivariate analyses, the aspects of applications significantly associated with composite Milestone scores at the end of internship were letter of recommendation strength (estimate 0.09, 95% confidence intervals [CI]: 0.04, 0.15), numbers of clerkship honors (est. 0.05, 95% CI: 0.01–0.09), medical school ranking (est. 0.04, 95% CI: 0.08–0.01), having a master's degree (est. 0.19, 95% CI: 0.03–0.36), and not having a PhD (est. 0.14, 95% CI: 0.02–0.26). Overall, the final model explained 18% of the variance in milestone scoring. Letter of recommendation strength, clerkship grades, medical school ranking, and having obtained a Master's degree were significantly associated with higher clinical performance during pediatric internship. [ABSTRACT FROM AUTHOR]

The ternary complex of spectrin, F-actin and protein 4.1R binds the N-terminal region of the spectrin chain containing two calponin homology domains CH1 and CH2. It is observed that the formation and binding of spectrin-actin-4.1R ternary complex is inhibited by the polypeptide 1-301 and this binding acts like a regulatory switch in the cell.

Whole-exome sequencing is increasingly used for diagnosis and identification of appropriate therapies in patients. Here, we present the case of a 3-yr-old male with a lifelong and severe transfusion-dependent anemia of unclear etiology, despite an extensive clinical workup. Given the difficulty of making the diagnosis and the potential side effects from performing interventions in patients with a congenital anemia of unknown etiology, we opted to perform whole-exome sequencing on the patient and his parents. This resulted in the identification of homozygous loss-of-function mutations in the EPB41 gene, encoding erythrocyte protein band 4.1, which therefore causes a rare and severe form of hereditary elliptocytosis in the patient. Based on prior clinical experience in similar patients, a surgical splenectomy was performed that resulted in subsequent transfusion independence in the patient. This case illustrates how whole-exome sequencing can lead to accurate diagnoses (and exclusion of diagnoses where interventions, such as splenectomy, would be contraindicated), thereby resulting in appropriate and successful therapeutic intervention—a major goal of precision medicine.

Four patients with overhydrated cation leak stomatocytosis (OHSt) exhibited the heterozygous RhAG missense mutation F65S. OHSt erythrocytes were osmotically fragile, with elevated Na and decreased K contents and increased cation channel-like activity. Xenopus oocytes expressing wildtype RhAG and RhAG F65S exhibited increased ouabain and bumetanide-resistant uptake of Li+ and 86Rb+, with secondarily increased 86Rb+ influx sensitive to ouabain and to bumetanide. Increased RhAG-associated 14C-methylammonium (MA) influx was severely reduced in RhAG F65S-expressing oocytes. RhAG-associated influxes of Li+, 86Rb+, and 14C-MA were pharmacologically distinct, and Li+ uptakes associated with RhAG and RhAG F65S were differentially inhibited by NH4+ and Gd3+. RhAG-expressing oocytes were acidified and depolarized by 5 mM bath NH3/NH4+, but alkalinized and depolarized by subsequent bath exposure to 5 mM methylammonium chloride (MA/MA+). RhAG F65S-expressing oocytes exhibited near-wildtype responses to NH4Cl, but MA/MA+ elicited attenuated alkalinization and strong hyperpolarization. Expression of RhAG or RhAG F65S increased steady-state cation currents unaltered by bath Li+ substitution or bath addition of 5 mM NH4Cl or MA/MA+. These oocyte studies suggest that 1) RhAG expression increases oocyte transport of NH3/NH4+ and MA/MA+; 2) RhAG F65S exhibits gain-of-function phenotypes of increased cation conductance/permeability, and loss-of-function phenotypes of decreased and modified MA/MA+ transport, and decreased NH3/NH4+ -associated depolarization; 3) RhAG transports NH3/NH4+ and MA/MA+ by distinct mechanisms, and/or the substrates elicit distinct cellular responses. Thus, RhAG F65S is a loss-of-function mutation for amine transport. The altered oocyte pHi, Vm, and currents associated with RhAG or RhAG F65S expression may reflect distinct transport mechanisms. [ABSTRACT FROM AUTHOR]

Summary. Nonsense/stop mutations in the ankyrin-1 gene (ANK1 ) are a major cause of dominant HS (dHS) (frequency of 23% in German dHS patients). To date, no common mutation has been found and therefore a simple mutation screening is not feasible. The reduced expression of one cDNA allele in the (AC)n microsatellite polymorphism of the ankyrin-1 gene, as seen in about 20% of Czech patients with dHS, may identify candidates with a possible frameshift/nonsense mutation. In order to verify the efficiency of this screening we screened the ankyrin-1 gene of 22 Czech dHS patients for both the reduced cDNA allele expression in the frequent (AC)n and the common exonic 26/39 polymorphisms, as well as for polymerase chain reaction (PCR) single-stranded conformation polymorphisms in any one of the 42 exons of ANK1 . Anomalous PCR products were sequenced. We found seven new ANK1 frameshift/nonsense mutations in nine patients with, but in none of six patients without, a reduced cDNA allele expression (efficiency of 78%). We conclude that screening of dHS patients for such a reduced allele expression in common ANK1 polymorphisms is an efficient procedure for the identification of candidates for frameshift/nonsense mutations in the ankyrin-1 gene. [ABSTRACT FROM AUTHOR]

The binary interaction between human erythrocyte protein 4.1 and rabbit skeletal muscle F-actin was examined by rapid pelleting of the binary complexes. The binding curves show that the reaction was saturable at approximately one protein 4.1 molecule/actin monomer. The reaction was highly co-operative, displaying a Hill coefficient close to 2. Using a fixed concentration of radiolabelled protein 4.1, and varying the concentration of F-actin, the apparent molar association constant, Ka, was observed to range from 5×104 M-1 to > 106 M-1. The binary interaction between erythrocyte spectrin and actin was also observed to be co-operative under the same conditions. The rate of reaction between protein 4.1 and actin was temperature sensitive in a manner consistent with a high energy of activation. The pelleting assay also showed that the concentration of actin was reduced in the supernatant in the presence of protein 4.1 compared with actin alone, indicating that the critical concentration of actin was lowered in the presence of protein 4.1. Polyvalent anions disrupted the binary interaction between F-actin and protein 4.1, the disruption being consistent with the number of negative charges on these anions at pH 7.5. We postulate that the co-operativity of the binding of protein 4.1 to actin results from a protein 4.1 molecule binding to a single monomer within the filament structure which then promotes conformational changes allowing further protein 4.1 binding. The demonstration of a specific binary association between protein 4.1 and actin suggests that this interaction contributes significantly to the stabilization of the spectrin—actin—protein-4.1 ternary complex. [ABSTRACT FROM AUTHOR]

Erythrocyte protein 4.1 plays a major role in stabilizing the spectrin-actin junction of the erythrocyte membrane skeleton. The particular sites on spectrin responsible for the binding of actin and protein 4.1 have not been specifically defined, although the general region of the ‘tail’ end, opposite the self-association site, has been deduced by electron microscopy. Using a photoactivatable, radiolabel-transfer cross-linker, 1-[N-(2-hydroxy-5-azidobenzoyl)-2-aminoethyl]-4-(N-hydroxysuccinimidyl)-succinate, we have determined that the binding site for protein 4.1 on spectrin resides in the N-terminal region of Β spectrin within a sequence homologous to the actin-binding region of α actinin. Moreover, this technique provided clear evidence for a direct binding interaction between actin filaments and protein 4.1 that was confirmed by rapid-sedimentation assays. In summary, use of radiolabel-transfer cross-linking has enabled assignment of the protein-4.1-binding site on erythrocyte spectrin and has identified a previously ill-defined binary interaction between protein 4.1 and F-actin. [ABSTRACT FROM AUTHOR]

Examines the distribution of epithelial ankyrin (Ank3) spliceoforms in proximal and distal renal tubules. Functions of the renal ankyrin cells; Ankyrin genes that have been cloned and sequenced; Methodology of the study; Specificity and functions of ank3 spliceoforms in the renal epithelial cells.

Spectrin and protein 4.1 cross-link F-actin protofilaments into a network called the membrane skeleton. Actin and 4.1 bind to one end of β-spectrin. The adjacent end of α-spectrin, called the EF-domain, is calmodulin-like, with calcium-dependent and calcium-independent EF-hands. It has no known function. However, the sph1J/sph1J mouse has very fragile red cells and lacks the last 13 amino acids in the EF-domain, suggesting the domain is critical for skeletal integrity. Using pulldown binding assays, we find the α-spectrin EF-domain either alone or incorporated into a mini-spectrin binds native and recombinant protein 4.2 at a previously identified region of 4.2 (G3 peptide). Native 4.2 binds with an affinity comparable with other membrane skeletal interactions (Kd = 0.30 ELM). EF-domains bearing the sph1J mutation are inactive. Binding of protein 4.2 to band 3 (Kd = 0.45 μM) does not interfere with the spectrin-4.2 interaction. Spectrin-4.2 binding is amplified by micromolar concentrations of Ca2+ (but not Mg2+) by three to five times. Calmodulin also binds to the EF-domain (Kd = 17 LM), and Ca2+-calmodulin blocks Ca2+-dependent binding of protein 4.2 but not Ca2+-independent binding. The data suggest that protein 4.2 is located near protein 4.1 at the spectrin-actin junctions. Because proteins 4.1 and 4.2 also bind to band 3, the erythrocyte anion channel, we suggest that one or both of these proteins cause a portion of band 3 to localize near the spectrin-actin junctions and provide another point of attachment between the membrane skeleton and the lipid bilayer. [ABSTRACT FROM AUTHOR]

The ternary complex of spectrin, F-actin, and protein 4.1R defines the erythrocyte membrane skeletal network, which governs the stability and elasticity of the membrane. It has been shown that both 4.1R and actin bind to the N-terminal region (residues 1-301) of the spectrin beta chain, which contains two calponin homology domains, designated CH1 and CH2. Here, we show that 4.1R also binds to the separate CH1 and CH2 domains. Unexpectedly, truncation of the CH2 domain by its 20 amino acids, corresponding to its N-terminal alpha helix, was found to greatly enhance its binding to 4.1R. The intact N terminus and the CH1 but not the CH2 domain bind to F-actin, but again, deletion of the first 20 amino acids of the latter exposes an actin-binding activity. As expected, the polypeptide 1-301 inhibits the binding of spectrin dimer to actin and formation of the spectrin-actin-4.1R ternary complex in vitro. Furthermore, the binding of 4.1R to 1-301 is greatly enhanced by PIP(2), implying the existence of a regulatory switch in the cell.