PTPN12 is an important tumor suppressor that plays critical roles in various physiological processes. However, the molecular basis underlying the substrate specificity of PTPN12 remains uncertain. Here, enzymological and crystallographic studies have enabled us to identify two distinct structural features that are crucial determinants of PTPN12 substrate specificity: the pY+1 site binding pocket and specific basic charged residues along its surface loops. Key structurally plastic regions and specific residues in PTPN12 enabled recognition of different HER2 phosphorylation sites and regulated specific PTPN12 functions. In addition, the structure of PTPN12 revealed a CDK2 phosphorylation site in a specific PTPN12 loop. Taken together, our results not only provide the working mechanisms of PTPN12 for desphosphorylation of its substrates but will also help in designing specific inhibitors of PTPN12.

TREK-1 is a member of the two-pore domain potassium channel family that is known as a leak channel and plays a key role in many physiological and pathological processes. The conformational transition of the selectivity filter is considered as an effective strategy for potassium channels to control the course of potassium efflux. It is well known that TREK-1 is regulated by a large volume of extracellular and intracellular signals. However, until now, little was known about the selectivity filter gating mechanism of the channel. In this research, it was found that Ba 2؉ blocked the TREK-1 channel in a concentration-and timedependent manner. A mutagenesis analysis showed that overlapped binding of Ba 2؉ at the assumed K ؉ binding site 4 (S4) within the selectivity filter was responsible for the inhibitory effects on TREK-1. Then, Ba 2؉ was used as a probe to explore the conformational transition in the selectivity filter of the channel. It was confirmed that collapsed conformations were induced by extracellular K ؉ -free and acidification at the selectivity filters, leading to nonconductive to permeable ions. Further detailed characterization demonstrated that the two conformations presented different properties. Additionally, the N-terminal truncated isoform (⌬N41), a product derived from alternative translation initiation, was identified as a constitutively nonconductive variant. Together, these results illustrate the important role of selectivity filter gating in the regulation of TREK-1 by the extracellular K ؉ and proton.Potassium channels are ubiquitous pore-forming transmembrane proteins that transport K ϩ ions selectively and rapidly across the biological membranes. The efflux of K ϩ ions is controlled not only by the electrochemical gradient, but also by the gating mechanism. Along the ion conduction pathway of potassium channels, three structures are arranged from intercellular to extracellular: the lower activation gate, the selectivity filter (SF) 2 , and the upper inactivation gate (also termed the C-type inactivation gate). Correspondingly, there are mainly two kinds of mechanisms controlling K ϩ ion passage. Manipulation of the lower activation gate controls the transition between the open and close state of the channel. The upper inactivation gate, which is characterized by slow kinetics, controls the transition between conduction and nonconduction of the pore. The selectivity filter of the K ϩ channels, formed by the highly conserved sequence TV(I)GY(F)G, plays a pivotal role in both mechanisms. Accumulating evidence shows that the selectivity filter itself has the ability to act as the inactivation gate (1-4). High resolution crystallographic analysis has revealed detailed structural changes in the selectivity filter associated with the activation gating and inactivation gating (5, 6). The carbonyl oxygens together with the side chain hydroxyl oxygen of the threonine define four equally spaced ion-binding sites that are commonly termed S1-S4, from the extracellular to the intracellular region (7)...

The striatum is the most prominent nucleus in the basal ganglia and plays an important role in motor movement regulation. The cholinergic interneurons (ChIs) in striatum are involved in the motion regulation by releasing acetylcholine (ACh) and modulating the output of striatal projection neurons. Here, we report that muscarinic ACh receptor (M receptor) agonists, ACh and Oxotremorine (OXO-M), decreased the firing frequency of ChIs by blocking the hyperpolarization-activated cyclic nucleotide-gated (HCN) channels. Scopolamine (SCO), a nonselective antagonist of M receptors, abolished the inhibition. OXO-M exerted its function by activating the Gi/o cAMP signaling cascade. The single-cell reverse transcription polymerase chain reaction (scRT-PCR) revealed that all the five subtypes of M receptors and four subtypes of HCN channels were expressed on ChIs. Among them, M2 receptors and HCN2 channels were the most dominant ones and expressed in every single studied cholinergic interneuron (ChI).Our results suggest that ACh regulates not only the output of striatal projection neurons, but also the firing activity of ChIs themselves by activating presynaptic M receptors in the dorsal striatum. The activation of M2 receptors and blockage of HCN2 channels may play an important role in ACh inhibition on the excitability of ChIs. This finding adds a new G-protein coupled receptor mediated regulation on ChIs and provides a cellular mechanism for control of cholinergic activity and ACh release in the dorsal striatum.

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TREK-2, a member of two-pore-domain potassium channel family, regulates cellular excitability in response to diverse stimuli. However, how such stimuli control channel function remains unclear. Here, by characterizing the responses of cytosolic proximal C-terminus deletant (ΔpCt) and transmembrane segment 4 (M4)-glycine hinge mutant (G312A) to 2-Aminoethoxydiphenyl borate (2-APB), an activator of TREK-2, we show that the transduction initiated from pCt domain is allosterically coupled with the conformation of selectivity filter (SF) via the movements of M4, without depending on the original status of SF. Moreover, ΔpCt and G312A also exhibited blunted responses to extracellular alkalization, a model to induce SF conformational transition. These results suggest that the coupling between pCt domain and SF is bidirectional, and M4 movements are involved in both processes. Further mechanistic exploration reveals that the function of Phe316, a residue close to the C-terminus of M4, is associated with such communications. However, unlike TREK-2, M4-hinge of TREK-1 only controls the transmission from pCt to SF, rather than SF conformational changes triggered by pHo changes. Together, our findings uncover the unique gating properties of TREK-2, and elucidate the mechanisms for how the extracellular and intracellular stimuli harness the pore gating allosterically.