Cryo-EM structures of pannexin 1 and 3 reveal differences among pannexin isoforms.

Pannexins are single-membrane large-pore channels that release ions and ATP upon activation. Three isoforms of pannexins 1, 2, and 3, perform diverse cellular roles and differ in their pore lining residues. In this study, we report the cryo-EM structure of pannexin 3 at 3.9 Å and analyze its structural differences with pannexin isoforms 1 and 2. The pannexin 3 vestibule has two distinct chambers and a wider pore radius in comparison to pannexins 1 and 2. We further report two cryo-EM structures of pannexin 1, with pore substitutions W74R/R75D that mimic the pore lining residues of pannexin 2 and a germline mutant of pannexin 1, R217H at resolutions of 3.2 Å and 3.9 Å, respectively. Substitution of cationic residues in the vestibule of pannexin 1 results in reduced ATP interaction propensities to the channel. The germline mutant R217H in transmembrane helix 3 (TM3), leads to a partially constricted pore, reduced ATP interaction and weakened voltage sensitivity. The study compares the three pannexin isoform structures, the effects of substitutions of pore and vestibule-lining residues and allosteric effects of a pathological substitution on channel structure and function thereby enhancing our understanding of this vital group of ATP-release channels.

Mechanisms of neurotransmitter transport and drug inhibition in human VMAT2.

Monoamine neurotransmitters such as dopamine and serotonin control important brain pathways, including movement, sleep, reward and mood1. Dysfunction of monoaminergic circuits has been implicated in various neurodegenerative and neuropsychiatric disorders2. Vesicular monoamine transporters (VMATs) pack monoamines into vesicles for synaptic release and are essential to neurotransmission3-5. VMATs are also therapeutic drug targets for a number of different conditions6-9. Despite the importance of these transporters, the mechanisms of substrate transport and drug inhibition of VMATs have remained elusive. Here we report cryo-electron microscopy structures of the human vesicular monoamine transporter VMAT2 in complex with the antichorea drug tetrabenazine, the antihypertensive drug reserpine or the substrate serotonin. Remarkably, the two drugs use completely distinct inhibition mechanisms. Tetrabenazine binds VMAT2 in a lumen-facing conformation, locking the luminal gating lid in an occluded state to arrest the transport cycle. By contrast, reserpine binds in a cytoplasm-facing conformation, expanding the vestibule and blocking substrate access. Structural analyses of VMAT2 also reveal the conformational changes following transporter isomerization that drive substrate transport into the vesicle. These findings provide a structural framework for understanding the physiology and pharmacology of neurotransmitter packaging by synaptic vesicular transporters.

Structural basis of norepinephrine recognition and transport inhibition in neurotransmitter transporters.

Norepinephrine is a biogenic amine neurotransmitter that has widespread effects on alertness, arousal and pain sensation. Consequently, blockers of norepinephrine uptake have served as vital tools to treat depression and chronic pain. Here, we employ the Drosophila melanogaster dopamine transporter as a surrogate for the norepinephrine transporter and determine X-ray structures of the transporter in its substrate-free and norepinephrine-bound forms. We also report structures of the transporter in complex with inhibitors of chronic pain including duloxetine, milnacipran and a synthetic opioid, tramadol. When compared to dopamine, we observe that norepinephrine binds in a different pose, in the vicinity of subsite C within the primary binding site. Our experiments reveal that this region is the binding site for chronic pain inhibitors and a determinant for norepinephrine-specific reuptake inhibition, thereby providing a paradigm for the design of specific inhibitors for catecholamine neurotransmitter transporters.

Structure and Gating Dynamics of Na+/Cl- Coupled Neurotransmitter Transporters.

Neurotransmitters released at the neural synapse through vesicle exocytosis are spatiotemporally controlled by the action of neurotransmitter transporters. Integral membrane proteins of the solute carrier 6 (SLC6) family are involved in the sodium and chloride coupled uptake of biogenic amine neurotransmitters including dopamine, serotonin, noradrenaline and inhibitory neurotransmitters including glycine and γ-amino butyric acid. This ion-coupled symport works through a well-orchestrated gating of substrate through alternating-access, which is mediated through movements of helices that resemble a rocking-bundle. A large array of commercially prescribed drugs and psychostimulants selectively target neurotransmitter transporters thereby modulating their levels in the synaptic space. Drug-induced changes in the synaptic neurotransmitter levels can be used to treat depression or neuropathic pain whereas in some instances prolonged usage can lead to habituation. Earlier structural studies of bacterial neurotransmitter transporter homolog LeuT and recent structure elucidation of the Drosophila dopamine transporter (dDAT) and human serotonin transporter (hSERT) have yielded a wealth of information in understanding the transport and inhibition mechanism of neurotransmitter transporters. Computational studies based on the structures of dDAT and hSERT have shed light on the dynamics of varied components of these molecular gates in affecting the uphill transport of neurotransmitters. This review seeks to address structural dynamics of neurotransmitter transporters at the extracellular and intracellular gates and the effect of inhibitors on the ligand-binding pocket. We also delve into the effect of additional factors including lipids and cytosolic domains that influence the translocation of neurotransmitters across the membrane.