The tetraspan LHFPL5 is critical to establish maximal force sensitivity of the mechanotransduction channel of cochlear hair cells.

The mechanoelectrical transduction (MET) channel of cochlear hair cells is gated by the tip link, but the mechanisms that establish the exquisite force sensitivity of this MET channel are not known. Here, we show that the tetraspan lipoma HMGIC fusion partner-like 5 (LHFPL5) directly couples the tip link to the MET channel. Disruption of these interactions severely perturbs MET. Notably, the N-terminal cytoplasmic domain of LHFPL5 binds to an amphipathic helix in TMC1, a critical gating domain conserved between different MET channels. Mutations in the amphipathic helix of TMC1 or in the N-terminus of LHFPL5 that perturb interactions of LHFPL5 with the amphipathic helix affect channel responses to mechanical force. We conclude that LHFPL5 couples the tip link to the MET channel and that channel gating depends on a structural element in TMC1 that is evolutionarily conserved between MET channels. Overall, our findings support a tether model for transduction channel gating by the tip link.

Sensing sound: Cellular specializations and molecular force sensors.

Organisms of all phyla express mechanosensitive ion channels with a wide range of physiological functions. In recent years, several classes of mechanically gated ion channels have been identified. Some of these ion channels are intrinsically mechanosensitive. Others depend on accessory proteins to regulate their response to mechanical force. The mechanotransduction machinery of cochlear hair cells provides a particularly striking example of a complex force-sensing machine. This molecular ensemble is embedded into a specialized cellular compartment that is crucial for its function. Notably, mechanotransduction channels of cochlear hair cells are not only critical for auditory perception. They also shape their cellular environment and regulate the development of auditory circuitry. Here, we summarize recent discoveries that have shed light on the composition of the mechanotransduction machinery of cochlear hair cells and how this machinery contributes to the development and function of the auditory system.

CIB2 and CIB3 are auxiliary subunits of the mechanotransduction channel of hair cells.

CIB2 is a Ca2+- and Mg2+-binding protein essential for mechanoelectrical transduction (MET) by cochlear hair cells, but not by vestibular hair cells that co-express CIB2 and CIB3. Here, we show that in cochlear hair cells, CIB3 can functionally substitute for CIB2. Using X-ray crystallography, we demonstrate that CIB2 and CIB3 are structurally similar to KChIP proteins, auxiliary subunits of voltage-gated Kv4 channels. CIB2 and CIB3 bind to TMC1/2 through a domain in TMC1/2 flanked by transmembrane domains 2 and 3. The co-crystal structure of the CIB-binding domain in TMC1 with CIB3 reveals that interactions are mediated through a conserved CIB hydrophobic groove, similar to KChIP1 binding of Kv4. Functional studies in mice show that CIB2 regulates TMC1/2 localization and function in hair cells, processes that are affected by deafness-causing CIB2 mutations. We conclude that CIB2 and CIB3 are MET channel auxiliary subunits with striking similarity to Kv4 channel auxiliary subunits.

TMIE Defines Pore and Gating Properties of the Mechanotransduction Channel of Mammalian Cochlear Hair Cells.

TMC1 and TMC2 (TMC1/2) have been proposed to form the pore of the mechanotransduction channel of cochlear hair cells. Here, we show that TMC1/2 cannot form mechanotransduction channels in cochlear hair cells without TMIE. TMIE binds to TMC1/2, and a TMIE mutation that perturbs TMC1/2 binding abolishes mechanotransduction. N-terminal TMIE deletions affect the response of the mechanotransduction channel to mechanical force. Similar to mechanically gated TREK channels, the C-terminal cytoplasmic TMIE domain contains charged amino acids that mediate binding to phospholipids, including PIP2. TMIE point mutations in the C terminus that are linked to deafness disrupt phospholipid binding, sensitize the channel to PIP2 depletion from hair cells, and alter the channel's unitary conductance and ion selectivity. We conclude that TMIE is a subunit of the cochlear mechanotransduction channel and that channel function is regulated by a phospholipid-sensing domain in TMIE with similarity to those in other mechanically gated ion channels.

Mechanotransduction by PCDH15 Relies on a Novel cis-Dimeric Architecture.

The tip link, a filament formed by protocadherin 15 (PCDH15) and cadherin 23, conveys mechanical force from sound waves and head movement to open hair-cell mechanotransduction channels. Tip-link cadherins are thought to have acquired structural features critical for their role in mechanotransduction. Here, we biophysically and structurally characterize the unusual cis-homodimeric architecture of PCDH15. We show that PCDH15 molecules form double-helical assemblies through cis-dimerization interfaces in the extracellular cadherin EC2-EC3 domain region and in a unique membrane-proximal domain. Electron microscopy studies visualize the cis-dimeric PCDH15 assembly and reveal the PCDH15 extracellular domain as a parallel double helix with cis cross-bridges at the two locations we defined. The helical configuration suggests the potential for elasticity through helix winding and unwinding. Functional studies in hair cells show that mutations that perturb PCDH15 dimerization contacts affect mechanotransduction. Together, these data reveal the cis-dimeric architecture of PCDH15 and show that dimerization is critical for sensing mechanical stimuli.

Mechanically Gated Ion Channels in Mammalian Hair Cells.

Hair cells in the inner ear convert mechanical stimuli provided by sound waves and head movements into electrical signal. Several mechanically evoked ionic currents with different properties have been recorded in hair cells. The search for the proteins that form the underlying ion channels is still in progress. The mechanoelectrical transduction (MET) channel near the tips of stereociliary in hair cells, which is responsible for sensory transduction, has been studied most extensively. Several components of the sensory mechanotransduction machinery in stereocilia have been identified, including the multi-transmembrane proteins tetraspan membrane protein in hair cell stereocilia (TMHS)/LHFPL5, transmembrane inner ear (TMIE) and transmembrane channel-like proteins 1 and 2 (TMC1/2). However, there remains considerable uncertainty regarding the molecules that form the channel pore. In addition to the sensory MET channel, hair cells express the mechanically gated ion channel PIEZO2, which is localized near the base of stereocilia and not essential for sensory transduction. The function of PIEZO2 in hair cells is not entirely clear but it might have a role in damage sensing and repair processes. Additional stretch-activated channels of unknown molecular identity and function have been found to localize at the basolateral membrane of hair cells. Here, we review current knowledge regarding the different mechanically gated ion channels in hair cells and discuss open questions concerning their molecular composition and function.

Quantitative analysis of vesicle recycling at the calyx of Held synapse.

Vesicle recycling is pivotal for maintaining reliable synaptic signaling, but its basic properties remain poorly understood. Here, we developed an approach to quantitatively analyze the kinetics of vesicle recycling with exquisite signal and temporal resolution at the calyx of Held synapse. The combination of this electrophysiological approach with electron microscopy revealed that ∼80% of vesicles (∼270,000 out of ∼330,000) in the nerve terminal are involved in recycling. Under sustained stimulation, recycled vesicles start to be reused in tens of seconds when ∼47% of the preserved vesicles in the recycling pool (RP) are depleted. The heterogeneity of vesicle recycling as well as two kinetic components of RP depletion revealed the existence of a replenishable pool of vesicles before the priming stage and led to a realistic kinetic model that assesses the size of the subpools of the RP. Thus, our study quantified the kinetics of vesicle recycling and kinetically dissected the whole vesicle pool in the calyceal terminal into the readily releasable pool (∼0.6%), the readily priming pool (∼46%), the premature pool (∼33%), and the resting pool (∼20%).

Bulk endocytosis at neuronal synapses.

Neurotransmitter-containing synaptic vesicle (SV) fusion with the nerve terminal plasma membrane initiates neurotransmission in response to neuronal excitation. Under mild stimulation, the fused vesicular membrane is retrieved via kiss-and-run and/or clathrin-mediated endocytosis, which is sufficient to maintain recycling of SVs. When neurons are challenged with very high stimulation, the number of fused SVs can be extremely high, resulting in significant plasma membrane addition. Under such conditions, a higher capacity retrieval pathway, bulk endocytosis, is activated to redress this large membrane imbalance. Despite first being described more than 40 years ago, the molecular mechanisms underpinning this important process have yet to be clearly defined. In this review, we highlight the current evidence for bulk endocytosis and its prevalence in various neuronal models, as well as discuss the underlying molecular components.