Dysferlin is a large membrane protein involved in calcium-triggered resealing of the sarcolemma after injury. Although it is generally accepted that dysferlin is Ca(2+) sensitive, the Ca(2+) binding properties of dysferlin have not been characterized. In this study, we report an analysis of the Ca(2+) and membrane binding properties of all seven C2 domains of dysferlin as well as a multi-C2 domain construct. Isothermal titration calorimetry measurements indicate that all seven dysferlin C2 domains interact with Ca(2+) with a wide range of binding affinities. The C2A and C2C domains were determined to be the most sensitive, with Kd values in the tens of micromolar, whereas the C2D domain was least sensitive, with a near millimolar Kd value. Mutagenesis of C2A demonstrates the requirement for negatively charged residues in the loop regions for divalent ion binding. Furthermore, dysferlin displayed significantly lower binding affinity for the divalent cations magnesium and strontium. Measurement of a multidomain construct indicates that the solution binding affinity does not change when C2 domains are linked. Finally, sedimentation assays suggest all seven C2 domains bind lipid membranes, and that Ca(2+) enhances but is not required for interaction. This report reveals for the first time, to our knowledge, that all dysferlin domains bind Ca(2+) albeit with varying affinity and stoichiometry.

Sensory hair cells convert mechanical motion into chemical signals.Otoferlin, a six-C2 domain transmembrane protein linked to deafness in humans, is hypothesized to play a role in exocytosis at hair cell ribbon synapses. To date, however, otoferlin has been studied almost exclusively in mouse models, and no rescue experiments have been reported. Here we describe the phenotype associated with morpholino-induced otoferlin knockdown in zebrafish and report the results of rescue experiments conducted with full-length and truncated forms of otoferlin. We found that expression of otoferlin occurs early in development and is restricted to hair cells and the midbrain. Immunofluorescence microscopy revealed localization to both apical and basolateral regions of hair cells. Knockdown of otoferlin resulted in hearing and balance defects, as well as locomotion deficiencies. Further, otoferlin morphants had uninflated swim bladders. Rescue experiments conducted with mouse otoferlin restored hearing, balance, and inflation of the swim bladder. Remarkably, truncated forms of otoferlin retaining the C-terminal C2F domain also rescued the otoferlin knockdown phenotype, while the individual N-terminal C2A domain did not. We conclude that otoferlin plays an evolutionarily conserved role in vertebrate hearing and that truncated forms of otoferlin can rescue hearing and balance.H air cells couple mechanical motion to neurotransmitter release at synapses (1). In contrast to conventional neural synapses, hair cell synapses release neurotransmitter continuously and, in a graded manner (2), possess synaptic ribbons (2-4), and lack synaptophysin (5), complexin (6-9), Munc13 (10), and the calcium sensors synaptotagmin I and II (11). In place of synaptotagmin, it is believed that otoferlin may confer calcium sensitivity to evoke neurotransmitter release (12, 13). Otoferlin is a six-C2 domain transmembrane protein expressed in inner, outer, and vestibular hair cells, as well as restricted regions of the brain (13-16). In humans, missense mutations in otoferlin have been linked to hearing loss (17,18), and biochemical studies have determined that otoferlin binds calcium and lipids (12,19), as well as membrane trafficking proteins (12,(20)(21)(22)(23). Further, in vitro assays have demonstrated that otoferlin accelerates SNARE-mediated membrane fusion (12). Based upon this evidence, it is hypothesized that otoferlin functions as a calcium-sensitive regulator of neurotransmitter release in sensory hair cells.However, the results of several studies have raised questions related to otoferlin's function. For instance, despite otoferlin expression in vestibular hair cells, knockout mice show no balance defects (24, 25) despite reduced exocytosis in vestibular type I hair cells (24,26). This raises questions as to the importance of otoferlin in this system. Further, otoferlin did not rescue synchronous neurotransmitter release in synaptotagmin I knockout cultured neurons, indicating that otoferlin and synaptotagmin are not functionally redundant (2...

Otoferlin is a transmembrane protein consisting of six C2 domains, proposed to act as a calcium sensor for exocytosis. Although otoferlin is believed to bind calcium and lipids, the lipid specificity and identity of the calcium binding domains are controversial. Further, it is currently unclear whether the calcium binding affinity of otoferlin quantitatively matches the maximal intracellular presynaptic calcium concentrations of ∼30–50 μM known to elicit exocytosis. To characterize the calcium and lipid binding properties of otoferlin, we used isothermal titration calorimetry (ITC), liposome sedimentation assays, and fluorescence spectroscopy. Analysis of ITC data indicates that with the exception of the C2A domain, the C2 domains of otoferlin bind multiple calcium ions with moderate (Kd = 25–95 μM) and low affinities (Kd = 400–700 μM) in solution. However, in the presence of liposomes, the calcium sensitivity of the domains increased by up to 10-fold. It was also determined that calcium enhanced liposome binding for domains C2B–C2E, whereas the C2F domain bound liposomes in a calcium-independent manner. Mutations that abrogate calcium binding in C2F do not disrupt liposome binding, supporting the conclusion that the interaction of the C2F domain with phosphatidylserine is calcium-independent. Further, domains C2C and C2F, not domains C2A, C2B, C2D, and C2E, bound phosphatidylinositol 4,5-bisphosphate 1,2-dioleoyl-sn-glycero-3-phospho(1′-myoinositol-4′,5′-bisphosphate) [PI(4,5)P2], which preferentially steered them toward liposomes harboring PI(4,5)P2. Remarkably, lysine mutations L478A and L480A in C2C selectively weaken the PI(4,5)P2 interaction while leaving phosphatidylserine binding unaffected. Finally, shifts in the emission spectra of an environmentally sensitive fluorescent unnatural amino acid indicate that the calcium binding loops of the C2F domain directly interact with the lipid bilayer of negatively charged liposomes in a calcium-independent manner. On the basis of these results, we propose that the C2F and C2C domains of otoferlin preferentially bind PI(4,5)P2 and that PI(4,5)P2 may serve to target otoferlin to the presynapse in a calcium-independent manner. This positioning would facilitate fast calcium-dependent exocytosis at the hair cell synapse.

Sensory hair cells rely on otoferlin as the calcium sensor for exocytosis and encoding of sound preferentially over the neuronal calcium sensor synaptotagmin. Although it is established that synaptotagmin cannot rescue the otoferlin KO phenotype, the large size and low solubility of otoferlin have prohibited direct biochemical comparisons that could establish functional differences between these two proteins. To address this challenge, we have developed a singlemolecule colocalization binding titration assay (smCoBRA) that can quantitatively characterize full-length otoferlin from mammalian cell lysate. Using smCoBRA, we found that, although both otoferlin and synaptotagmin bind membrane fusion SNARE proteins, only otoferlin interacts with the L-type calcium channel Cav1.3, showing a significant difference between the synaptic proteins. Furthermore, otoferlin was found capable of interacting with multiple SNARE and Cav1.3 proteins simultaneously, forming a heterooligomer complex. We also found that a deafness-causing missense mutation in otoferlin attenuates binding between otoferlin and Cav1.3, suggesting that deficiencies in this interaction may form the basis for otoferlin-related hearing loss. Based on our results, we propose a model in which otoferlin acts as a calcium-sensitive scaffolding protein, localizing SNARE proteins proximal to the calcium channel so as to synchronize calcium influx with membrane fusion. Our findings also provide a molecular-level explanation for the observation that synaptotagmin and otoferlin are not functionally redundant. This study also validates a generally applicable methodology for quantitatively characterizing large, multivalent membrane proteins.exocytosis | membrane fusion | calcium | otoferlin | calcium channel S ensory hair cells encode sound by converting mechanical motion into chemical signals. Hair cell synapses accommodate this unique functional demand via a set of adaptations that distinguish it from neuronal synapses, including reliance on the L-type calcium channel Cav1.3 in place of the P-and N-type calcium channels found at most neuronal synapses ( Fig. 1 A and B) (1-6). During maturation, hair cells also cease expression of the two C2 domain protein synaptotagmin I, which serves as the calcium sensor for neurotransmitter release at neuronal synapses (7). Although lacking synaptotagmin, mature hair cells express the six C2 domain protein otoferlin, which is proposed to function as the calcium sensor for exocytosis in mature sound-encoding synapses (8). In agreement with this belief, KO studies have linked otoferlin to sensory hair cell exocytosis, and in vitro studies on otoferlin have concluded that several C2 domains bind SNARE proteins and stimulate membrane fusion in a calcium-sensitive manner (2,(8)(9)(10). However, a recent cell-based study concluded that synaptotagmin cannot rescue the otoferlin KO phenotype, arguing against a simple functional redundancy between these C2 domain proteins (11). The functional differences between synaptotagmin and otofer...

Proteins that contain C2 domains are involved in a variety of biological processes, including encoding of sound, cell signaling, and cell membrane repair. Of particular importance is the interface activity of the C-terminal C2F domain of otoferlin due to the pathological mutations known to significantly disrupt the protein's lipid membrane interface binding activity, resulting in hearing loss. Therefore, there is a critical need to define the geometry and positions of functionally important sites and structures at the otoferlin-lipid membrane interface. Here, we describe the first in situ probe of the protein orientation of otoferlin's C2F domain interacting with a cell membrane surface. To identify this protein's orientation at the lipid interface, we applied sum frequency generation (SFG) vibrational spectroscopy and coupled it with simulated SFG spectra to observe and quantify the otoferlin C2F domain interacting with model lipid membranes. A model cell membrane was built with equal amounts of phosphatidylserine and phosphatidylcholine. SFG measurements of the lipids that make up the model membrane indicate a 62% increase in amplitude from the SFG signal near 2075 cm À1 upon protein interaction, suggesting domain-induced changes in the orientation of the lipids and possible membrane curvature. This increase is related to lipid ordering caused by the docking interaction of the otoferlin C2F domain. SFG spectra taken from the amide-I region contain features near 1630 and 1670 cm À1 related to the C2F domains beta-sandwich secondary structure, thus indicating that the domain binds in a specific orientation. By mapping the simulated SFG spectra to the experimentally collected SFG spectra, we found the C2F domain of otoferlin orients 22 normal to the lipid surface. This information allows us to map what portion of the domain directly interacts with the lipid membrane.