The proteins that form the permeation pathway of mechanosensory transduction channels in inner-ear hair cells have not been definitively identified. Genetic, anatomical, and physiological evidence support a role for transmembrane channel-like protein (TMC) 1 in hair cell sensory transduction, yet the molecular function of TMC proteins remains unclear. Here, we provide biochemical evidence suggesting TMC1 assembles as a dimer, along with structural and sequence analyses suggesting similarity to dimeric TMEM16 channels. To identify the pore region of TMC1, we used cysteine mutagenesis and expressed mutant TMC1 in hair cells of Tmc1/2-null mice. Cysteine-modification reagents rapidly and irreversibly altered permeation properties of mechanosensory transduction. We propose that TMC1 is structurally similar to TMEM16 channels and includes ten transmembrane domains with four domains, S4-S7, that line the channel pore. The data provide compelling evidence that TMC1 is a pore-forming component of sensory transduction channels in auditory and vestibular hair cells.

Glutamate transporters terminate neurotransmission by clearing synaptically released glutamate from the extracellular space, allowing repeated rounds of signaling and preventing glutamate-mediated excitotoxicity. Crystallographic studies on an archaeal homologue, GltPh, showed that distinct transport domains translocate substrates into the cytoplasm by moving across the membrane within a central trimerization scaffold. Here, we report direct observations of these 'elevator-like' transport domain motions in the context of reconstituted proteoliposomes and physiological ion gradients using single-molecule fluorescence resonance energy transfer (smFRET) imaging. We show that GltPh bearing two “humanizing” mutations exhibits markedly increased transport domain dynamics, which parallels an increased rate of substrate transport, thereby establishing a direct temporal relationship between transport domain motions and substrate uptake. Crystallographic and computational investigations reveal that these mutations favor structurally “unlocked” states with increased solvent occupancy at the interface between the transport domain and the trimeric scaffold.

Summary Glutamate transporters are integral membrane proteins that catalyze neurotransmitter uptake from the synaptic cleft into the cytoplasm of glial cells and neurons1. Their mechanism involves transitions between extracellular- (outward-) and intracellular- (inward-) facing conformations, whereby substrate binding sites become accessible to the opposite sides of the membrane2. This process has been proposed to entail trans-membrane movements of three discrete transport domains within a trimeric scaffold3. Using single-molecule fluorescence resonance energy transfer (smFRET) imaging4, we have directly observed large-scale transport domain movements in a bacterial homologue of glutamate transporters for the first time. We find that individual transport domains alternate between periods of quiescence and periods of rapid transitions, reminiscent of bursting patterns first recorded in single ion channels using patch-clamp methods5,6. We suggest that the switch to the dynamic mode in glutamate transporters is due to separation of the transport domain from the trimeric scaffold, which precedes domain movements across the bilayer. This spontaneous dislodging of the substrate-loaded transport domain is approximately 100-fold slower than subsequent trans-membrane movements and may be rate determining in the transport cycle.

Glutamate transporters are integral membrane proteins that catalyze neurotransmitter uptake from the synaptic cleft into the cytoplasm of glial cells and neurons 1 . Their mechanism involves transitions between extracellular-(outward-) and intracellular-(inward-) facing conformations, whereby substrate binding sites become accessible to the opposite sides of the membrane 2 . This process has been proposed to entail trans-membrane movements of three discrete transport domains within a trimeric scaffold 3 . Using single-molecule fluorescence resonance energy transfer (smFRET) imaging 4 , we have directly observed large-scale transport domain movements in a bacterial homologue of glutamate transporters for the first time. We find that individual transport domains alternate between periods of quiescence and periods of rapid transitions, reminiscent of bursting patterns first recorded in single ion channels using patch-clamp methods 5,6 . We suggest that the switch to the dynamic mode in glutamate transporters is due to separation of the transport domain from the trimeric scaffold, which precedes domain movements across the bilayer. This spontaneous dislodging of the substrate-loaded transport domain is approximately 100-fold slower than subsequent trans-membrane movements and may be rate determining in the transport cycle. Main TextIn the brain, glutamate mediates excitatory synaptic transmission, responsible for learning, memory formation and cognition 1,7 . Glutamate transporters are electrochemically driven pumps that maintain a low neurotransmitter background at glutamatergic synapses, allowing for repeated rounds of signaling and preventing excitotoxicity 8 . The sodium/aspartate symporter from Pyrococcus horikoshii, Glt Ph , is the only glutamate transporter homologue with known three-dimensional structures of both outward-and inward-facing states 3,9 . Correspondingly, this system has served as a valuable model for establishing the structural and dynamic underpinnings of the transport cycle [10][11][12] . Because Glt Ph originates from a hyper-thermophilic archaeon, it has a slow turnover time of ∼100 seconds at room Users may view, print, copy, download and text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use: