The molecular basis for the endocytosis of small R-SNAREs by the clathrin adaptor CALM.

SNAREs provide a large part of the specificity and energy needed for membrane fusion and, to do so, must be localized to their correct membranes. Here, we show that the R-SNAREs VAMP8, VAMP3, and VAMP2, which cycle between the plasma membrane and endosomes, bind directly to the ubiquitously expressed, PtdIns4,5P(2)-binding, endocytic clathrin adaptor CALM/PICALM. X-ray crystallography shows that the N-terminal halves of their SNARE motifs bind the CALM(ANTH) domain as helices in a manner that mimics SNARE complex formation. Mutation of residues in the CALM:SNARE interface inhibits binding in vitro and prevents R-SNARE endocytosis in vivo. Thus, CALM:R-SNARE interactions ensure that R-SNAREs, required for the fusion of endocytic clathrin-coated vesicles with endosomes and also for subsequent postendosomal trafficking, are sorted into endocytic vesicles. CALM's role in directing the endocytosis of small R-SNAREs may provide insight into the association of CALM/PICALM mutations with growth retardation, cognitive defects, and Alzheimer's disease.

A SNARE-adaptor interaction is a new mode of cargo recognition in clathrin-coated vesicles.

Soluble NSF attachment protein receptors (SNAREs) are type II transmembrane proteins that have critical roles in providing the specificity and energy for transport-vesicle fusion and must therefore be correctly partitioned between vesicle and organelle membranes. Like all other cargo, SNAREs need to be sorted into the forming vesicles by direct interaction with components of the vesicles' coats. Here we characterize the molecular details governing the sorting of a SNARE into clathrin-coated vesicles, namely the direct recognition of the three-helical bundle H(abc) domain of the mouse SNARE Vti1b by the human clathrin adaptor epsinR (EPNR, also known as CLINT1). Structures of each domain and of their complex show that this interaction (dissociation constant 22 muM) is mediated by surface patches composed of approximately 15 residues each, the topographies of which are dependent on each domain's overall fold. Disruption of the interface with point mutations abolishes the interaction in vitro and causes Vti1b to become relocalized to late endosomes and lysosomes. This new class of highly specific, surface-surface interaction between the clathrin coat component and the cargo is distinct from the widely observed binding of short, linear cargo motifs by the assembly polypeptide (AP) complex and GGA adaptors and is therefore not vulnerable to competition from standard motif-containing cargoes for incorporation into clathrin-coated vesicles. We propose that conceptually similar but mechanistically different interactions will direct the post-Golgi trafficking of many SNAREs.

Neural Mechanisms of the Acceptable Noise Level.

PURPOSE: The present brain-behavior study examined whether sensory registration or neural inhibition processes explained variability in the behavioral most comfortable level (MCL) and background noise level (BNL) components of the acceptable noise level (ANL) measure. METHOD: A traditional auditory gating paradigm was used to evoke neural responses to pairs of pure-tone stimuli in 32 adult listeners with normal hearing. Relationships between behavioral ANL, MCL, and BNL components and cortical responses to each of the paired stimuli were analyzed using linear mixed-effects regression analyses. RESULTS: Neural responses elicited by Stimulus 2 in the gating paradigm significantly predicted the computed ANL response. The MCL component was significantly associated with responses elicited by Stimulus 1 of the pair. The BNL component of the ANL was significantly associated with neural responses to both Stimulus 1 and Stimulus 2. CONCLUSIONS: The results suggest neural processes related to neural inhibition support the ANL and BNL component while neural stimulus registration properties are associated with the MCL a listener chooses. These findings suggest that differential neural mechanisms underlie the separate MCL and BNL components of the ANL response.

Auditory Sensory Gating of Speech and Nonspeech Stimuli.

Purpose Auditory sensory gating is a neural measure of inhibition and is typically measured with a click or tonal stimulus. This electrophysiological study examined if stimulus characteristics and the use of speech stimuli affected auditory sensory gating indices. Method Auditory event-related potentials were elicited using natural speech, synthetic speech, and nonspeech stimuli in a traditional auditory gating paradigm in 15 adult listeners with normal hearing. Cortical responses were recorded at 64 electrode sites, and peak amplitudes and latencies to the different stimuli were extracted. Individual data were analyzed using repeated-measures analysis of variance. Results Significant gating of P1-N1-P2 peaks was observed for all stimulus types. N1-P2 cortical responses were affected by stimulus type, with significantly less neural inhibition of the P2 response observed for natural speech compared to nonspeech and synthetic speech. Conclusions Auditory sensory gating responses can be measured using speech and nonspeech stimuli in listeners with normal hearing. The results of the study indicate the amount of gating and neural inhibition observed is affected by the spectrotemporal characteristics of the stimuli used to evoke the neural responses.

The effects of fundamental frequency contour manipulations on speech intelligibility in background noise.

Previous studies have documented that speech with flattened or inverted fundamental frequency (F0) contours is less intelligible than speech with natural variations in F0. The purpose of this present study was to further investigate how F0 manipulations affect speech intelligibility in background noise. Speech recognition in noise was measured for sentences having the following F0 contours: unmodified, flattened at the median, natural but exaggerated, inverted, and sinusoidally frequency modulated at rates of 2.5 and 5.0 Hz, rates shown to make vowels more perceptually salient in background noise. Five talkers produced 180 stimulus sentences, with 30 unique sentences per F0 contour condition. Flattening or exaggerating the F0 contour reduced key word recognition performance by 13% relative to the naturally produced speech. Inverting or sinusoidally frequency modulating the F0 contour reduced performance by 23% relative to typically produced speech. These results support the notion that linguistically incorrect or misleading cues have a greater deleterious effect on speech understanding than linguistically neutral cues.

Neural Coding of Syllable-Final Fricatives with and without Hearing Aid Amplification.

BACKGROUND: Cortical auditory event-related potentials are a potentially useful clinical tool to objectively assess speech outcomes with rehabilitative devices. Whether hearing aids reliably encode the spectrotemporal characteristics of fricative stimuli in different phonological contexts and whether these differences result in distinct neural responses with and without hearing aid amplification remain unclear. PURPOSE: To determine whether the neural coding of the voiceless fricatives /s/ and /ʃ/ in the syllable-final context reliably differed without hearing aid amplification and whether hearing aid amplification altered neural coding of the fricative contrast. RESEARCH DESIGN: A repeated-measures, within subject design was used to compare the neural coding of a fricative contrast with and without hearing aid amplification. STUDY SAMPLE: Ten adult listeners with normal hearing participated in the study. DATA COLLECTION AND ANALYSIS: Cortical auditory event-related potentials were elicited to an /ɑs/-/ɑʃ/ vowel-fricative contrast in unaided and aided listening conditions. Neural responses to the speech contrast were recorded at 64-electrode sites. Peak latencies and amplitudes of the cortical response waveforms to the fricatives were analyzed using repeated-measures analysis of variance. RESULTS: The P2' component of the acoustic change complex significantly differed from the syllable-final fricative contrast with and without hearing aid amplification. Hearing aid amplification differentially altered the neural coding of the contrast across frontal, temporal, and parietal electrode regions. CONCLUSIONS: Hearing aid amplification altered the neural coding of syllable-final fricatives. However, the contrast remained acoustically distinct in the aided and unaided conditions, and cortical responses to the fricative significantly differed with and without the hearing aid.

Insurance Payer Status Predicts Postoperative Speech Outcomes in Adult Cochlear Implant Recipients.

BACKGROUND: Cochlear implant qualifying criteria for adult patients with public insurance policies are stricter than the labeled manufacturer criteria. It remains unclear whether insurance payer status affects expedient access to implants for adult patients who could derive benefit from the devices. PURPOSE: This study examined whether insurance payer status affected access to cochlear implant services and longitudinal speech-perception outcomes in adult cochlear implant recipients. RESEARCH DESIGN: Retrospective cross-sectional study. STUDY SAMPLE: Sixty-eight data points were queried from the Health Insurance Portability and Accountability Act-Secure, Encrypted, Research Management and Evaluation Solution database which consists of 12,388 de-identified data points from adult and pediatric cochlear implant recipients. DATA ANALYSIS: Linear mixed-effects models were used to determine whether insurance payer status affected expedient access to cochlear implants and whether payer status predicted longitudinal postoperative speech-perception scores in quiet and noise. RESULTS: Results from linear mixed-effects regression models indicated that insurance payer status was a significant predictor of behavioral speech-perception scores in quiet and in background noise, with patients with public insurance experiencing poorer outcomes. In addition, extended wait time to receive a cochlear implant was predicted to significantly decrease speech-perception outcomes for patients with public insurance. CONCLUSION: This study documented patients covered by public health insurance wait longer to receive cochlear implants and experience poorer postoperative speech-perception outcomes. These results have important clinical implications regarding the cochlear implant candidacy criteria and intervention protocols.

Benefits of a Hearing Registry: Cochlear Implant Candidacy in Quiet Versus Noise in 1,611 Patients.

Purpose This retrospective study used a cochlear implant registry to determine how performing speech recognition candidacy testing in quiet versus noise influenced patient selection, speech recognition, and self-report outcomes. Method Database queries identified 1,611 cochlear implant recipients who were divided into three implant candidacy qualifying groups based on preoperative speech perception scores (≤ 40% correct) on the AzBio sentence test: quiet qualifying group, +10 dB SNR qualifying group, and +5 dB SNR qualifying group. These groups were evaluated for demographic and preoperative hearing characteristics. Repeated-measures analysis of variance was used to compare pre- and postoperative performance on the AzBio in quiet and noise with qualifying group as a between-subjects factor. For a subset of recipients, pre- to postoperative changes on the Speech, Spatial and Qualities of Hearing Scale were also evaluated. Results Of the 1,611 patients identified as cochlear implant candidates, 63% of recipients qualified in quiet, 10% qualified in a +10 dB SNR, and 27% qualified in a +5 dB SNR. Postoperative speech perception scores in quiet and noise significantly improved for all qualifying groups. Across qualifying groups, the greatest speech perception improvements were observed when tested in the same qualifying listening condition. For a subset of patients, the total Speech, Spatial and Qualities of Hearing Scale ratings improved significantly as well. Conclusion Patients who qualified for cochlear implantation in quiet or background noise test conditions showed significant improvement in speech perception and quality of life scores, especially when the qualifying noise condition was used to track performance.

EpsinR is an adaptor for the SNARE protein Vti1b.

EpsinR is a clathrin-coated vesicle (CCV)-associated protein that binds to vti1b, suggesting that it may be a vti1b-selective adaptor. Depletion of epsinR to undetectable levels in HeLa cells using siRNA causes vti1b to redistribute from the perinuclear region to the cell periphery, but vti1a also redistributes in epsinR-depleted cells, and both vti isoforms redistribute in AP-1-depleted cells. As a more direct assay for epsinR function, we isolated CCVs from control and siRNA-treated cells and then looked for differences in cargo content. In clathrin-depleted cells, both coat and cargo proteins are greatly reduced in this preparation. Knocking down epsinR causes a approximately 50% reduction in the amount of AP-1 copurifying with CCVs and vice versa, indicating that the two proteins are dependent on each other for maximum incorporation into the coat. In addition, vti1b, but not vti1a, is reduced by >70% in CCVs from both epsinR- and AP-1-depleted cells. Because AP-1 knockdown reduces the amount of epsinR in CCVs, it is possible that its effect on vti1b may be indirect. These findings provide in vivo evidence that epsinR is an adaptor for vti1b, and they also show that CCV isolation can be used as an assay for adaptor function.

Efficacy of Multiple-Talker Phonetic Identification Training in Postlingually Deafened Cochlear Implant Listeners.

PURPOSE: This study implemented a pretest-intervention-posttest design to examine whether multiple-talker identification training enhanced phonetic perception of the /ba/-/da/ and /wa/-/ja/ contrasts in adult listeners who were deafened postlingually and have cochlear implants (CIs). METHOD: Nine CI recipients completed 8 hours of identification training using a custom-designed training package. Perception of speech produced by familiar talkers (talkers used during training) and unfamiliar talkers (talkers not used during training) was measured before and after training. Five additional untrained CI recipients completed identical pre- and posttests over the same time course as the trainees to control for procedural learning effects. RESULTS: Perception of the speech contrasts produced by the familiar talkers significantly improved for the trained CI listeners, and effects of perceptual learning transferred to unfamiliar talkers. Such training-induced significant changes were not observed in the control group. CONCLUSION: The data provide initial evidence of the efficacy of the multiple-talker identification training paradigm for CI users who were deafened postlingually. This pattern of results is consistent with enhanced phonemic categorization of the trained speech sounds.

CALM regulates clathrin-coated vesicle size and maturation by directly sensing and driving membrane curvature.

The size of endocytic clathrin-coated vesicles (CCVs) is remarkably uniform, suggesting that it is optimized to achieve the appropriate levels of cargo and lipid internalization. The three most abundant proteins in mammalian endocytic CCVs are clathrin and the two cargo-selecting, clathrin adaptors, CALM and AP2. Here we demonstrate that depletion of CALM causes a substantial increase in the ratio of "open" clathrin-coated pits (CCPs) to "necked"/"closed" CCVs and a doubling of CCP/CCV diameter, whereas AP2 depletion has opposite effects. Depletion of either adaptor, however, significantly inhibits endocytosis of transferrin and epidermal growth factor. The phenotypic effects of CALM depletion can be rescued by re-expression of wild-type CALM, but not with CALM that lacks a functional N-terminal, membrane-inserting, curvature-sensing/driving amphipathic helix, the existence and properties of which are demonstrated. CALM is thus a major factor in controlling CCV size and maturation and hence in determining the rates of endocytic cargo uptake.

Uncoupling the functions of CALM in VAMP sorting and clathrin-coated pit formation.

CALM (clathrin assembly lymphoid myeloid leukemia protein) is a cargo-selective adaptor for the post-Golgi R-SNAREs VAMPs 2, 3, and 8, and it also regulates the size of clathrin-coated pits and vesicles at the plasma membrane. The present study has two objectives: to determine whether CALM can sort additional VAMPs, and to investigate whether VAMP sorting contributes to CALM-dependent vesicle size regulation. Using a flow cytometry-based endocytosis efficiency assay, we demonstrate that CALM is also able to sort VAMPs 4 and 7, even though they have sorting signals for other clathrin adaptors. CALM homologues are present in nearly every eukaryote, suggesting that the CALM family may have evolved as adaptors for retrieving all post-Golgi VAMPs from the plasma membrane. Using a knockdown/rescue system, we show that wild-type CALM restores normal VAMP sorting in CALM-depleted cells, but that two non-VAMP-binding mutants do not. However, when we assayed the effect of CALM depletion on coated pit morphology, using a fluorescence microscopy-based assay, we found that the two mutants were as effective as wild-type CALM. Thus, we can uncouple the sorting function of CALM from its structural role.