A new mechanism of voltage-dependent gating exposed by KV10.1 channels interrupted between voltage sensor and pore

Voltage-gated ion channels couple transmembrane potential changes to ion flow. Conformational changes in the voltage-sensing domain (VSD) of the channel are thought to be transmitted to the pore domain (PD) through an α-helical linker between them (S4-S5 linker). However, our recent work on channels disrupted in the S4-S5 linker has challenged this interpretation for the KCNH family. Furthermore, a recent single-particle cryo-electron microscopy structure of KV10.1 revealed that the S4-S5 linker is a short loop in this KCNH family member, confirming the need for an alternative gating model. Here we use "split" channels made by expression of VSD and PD as separate fragments to investigate the mechanism of gating in KV10.1. We find that disruption of the covalent connection within the S4 helix compromises the ability of channels to close at negative voltage, whereas disconnecting the S4-S5 linker from S5 slows down activation and deactivation kinetics. Surprisingly, voltage-clamp fluorometry and MTS accessibility assays show that the motion of the S4 voltage sensor is virtually unaffected when VSD and PD are not covalently bound. Finally, experiments using constitutively open PD mutants suggest that the presence of the VSD is structurally important for the conducting conformation of the pore. Collectively, our observations offer partial support to the gating model that assumes that an inward motion of the C-terminal S4 helix, rather than the S4-S5 linker, closes the channel gate, while also suggesting that control of the pore by the voltage sensor involves more than one mechanism

APP Homodimers Transduce an Amyloid-β-Mediated Increase in Release Probability at Excitatory Synapses

SummaryAccumulation of amyloid-β peptides (Aβ), the proteolytic products of the amyloid precursor protein (APP), induces a variety of synaptic dysfunctions ranging from hyperactivity to depression that are thought to cause cognitive decline in Alzheimer’s disease. While depression of synaptic transmission has been extensively studied, the mechanisms underlying synaptic hyperactivity remain unknown. Here, we show that Aβ40 monomers and dimers augment release probability through local fine-tuning of APP-APP interactions at excitatory hippocampal boutons. Aβ40 binds to the APP, increases the APP homodimer fraction at the plasma membrane, and promotes APP-APP interactions. The APP activation induces structural rearrangements in the APP/Gi/o-protein complex, boosting presynaptic calcium flux and vesicle release. The APP growth-factor-like domain (GFLD) mediates APP-APP conformational changes and presynaptic enhancement. Thus, the APP homodimer constitutes a presynaptic receptor that transduces signal from Aβ40 to glutamate release. Excessive APP activation may initiate a positive feedback loop, contributing to hippocampal hyperactivity in Alzheimer’s disease

Subunit composition of a DEG/ENaC mechanosensory channel of Caenorhabditis elegans

Caenorhabditis elegans senses gentle touch in the six touch receptor neurons (TRNs) using a mechanotransduction complex that contains the pore-forming degenerin/epithelial sodium channel (DEG/ENaC) proteins MEC-4 and MEC-10. Past work has suggested these proteins interact with the paraoxonase-like MEC-6 and the cholesterol-binding stomatin-like MEC-2 proteins. Using single molecule optical imaging in Xenopus oocytes, we found that MEC-4 forms homotrimers and MEC-4 and MEC-10 form 4:4:10 heterotrimers. MEC-6 and MEC-2 do not associate tightly with these trimers and do not influence trimer stoichiometry, indicating that they are not part of the core channel transduction complex. Consistent with the in vitro data, MEC-10, but not MEC-6, formed puncta in TRN neurites that colocalize with MEC-4 when MEC-4 is overexpressed in the TRNs

Subunit composition of a DEG/ENaC mechanosensory channel of Caenorhabditis elegans

Caenorhabditis elegans senses gentle touch in the six touch receptor neurons (TRNs) using a mechanotransduction complex that contains the pore-forming degenerin/epithelial sodium channel (DEG/ENaC) proteins MEC-4 and MEC-10. Past work has suggested these proteins interact with the paraoxonase-like MEC-6 and the cholesterol-binding stomatin-like MEC-2 proteins. Using single molecule optical imaging in Xenopus oocytes, we found that MEC-4 forms homotrimers and MEC-4 and MEC-10 form 4:4:10 heterotrimers. MEC-6 and MEC-2 do not associate tightly with these trimers and do not influence trimer stoichiometry, indicating that they are not part of the core channel transduction complex. Consistent with the in vitro data, MEC-10, but not MEC-6, formed puncta in TRN neurites that colocalize with MEC-4 when MEC-4 is overexpressed in the TRNs

Stoichiometry and specific assembly of Best ion channels

Human Bestrophin 1 (hBest1) is a calcium-activated chloride channel that regulates neuronal excitability, synaptic activity, and retinal homeostasis. Mutations in hBest1 cause the autosomal-dominant Best macular dystrophy (BMD). Because hBest1 mutations cause BMD, but a knockout does not, we wondered if hBest1 mutants exert a dominant negative effect through interaction with other calcium-activated chloride channels, such as hBest2, 3, or 4, or transmembrane member 16A (TMEM16A), a member of another channel family. The subunit architecture of Best channels is debated, and their ability to form heteromeric channel assemblies is unclear. Using single-molecule subunit analysis, we find that each of hBest1, 2, 3, and 4 forms a homotetrameric channel. Despite considerable conservation among hBests, hBest1 has little or no interaction with other hBests or mTMEM16A. We identify the domain responsible for assembly specificity. This domain also plays a role in channel function. Our results indicate that Best channels preferentially self-assemble into homotetramers

MEC-10 and MEC-19 Reduce the Neurotoxicity of the MEC-4(d) DEG/ENaC Channel in Caenorhabditis elegans.

The Caenorhabditis elegans DEG/ENaC proteins MEC-4 and MEC-10 transduce gentle touch in the six touch receptor neurons . Gain-of-function mutations of mec-4 and mec-4(d) result in a hyperactive channel and neurodegeneration in vivo Loss of MEC-6, a putative DEG/ENaC-specific chaperone, and of the similar protein POML-1 suppresses the neurodegeneration caused by a mec-4(d) mutation. We find that mutation of two genes, mec-10 and a new gene mec-19 (previously named C49G9.1), prevents this action of POML-1, allowing the touch receptor neurons to die in poml-1 mec-4(d) animals. The proteins encoded by these genes normally inhibit mec-4(d) neurotoxicity through different mechanisms. MEC-10, a subunit of the mechanosensory transduction channel with MEC-4, inhibits MEC-4(d) activity without affecting MEC-4 expression. In contrast, MEC-19, a membrane protein specific to nematodes, inhibits MEC-4(d) activity and reduces MEC-4 surface expression