Disruption Of Specific Interactions Research Articles

Engineering α4β2 nAChRs with reduced or increased nicotine sensitivity via selective disruption of consensus sites in the M3–M4 cytoplasmic loop of the α4 subunit

The α4β2 neuronal nicotinic acetylcholine receptor (nAChR) plays a crucial role in nicotine addiction. These receptors are known to desensitize and up-regulate after chronic nicotine exposure, but the mechanism remains unknown. Currently, the structure and functional role of the intracellular domains of the nAChR are obscure. To study the effect of subunit phosphorylation on α4β2 nAChR function and expression, eleven residues located in the M3–M4 cytoplasmic loop were mutated to alanine and aspartic acid. Two-electrode voltage clamp and 125I-labeled epibatidine binding assays were performed on Xenopus oocytes to assess agonist activation and receptor expression. When ACh was used as an agonist, a decrease in receptor activation was observed for the majority of the mutations. When nicotine was used as an agonist, four mutations exhibited a statistically significant hypersensitivity to nicotine (S438D, S469A, Y576A, and S589A). Additionally, two mutations (S516D and T536A) that displayed normal activation with ACh displayed remarkable reductions in sensitivity to nicotine. Binding assays revealed a constitutive up-regulation in these two nicotine mutations with reduced nicotine sensitivity. These results suggest that consensus phosphorylation residues in the M3–M4 cytoplasmic loop of the α4 subunit play a crucial role in regulating α4β2 nAChR agonist selectivity and functional expression. Furthermore, these results suggest that disruption of specific interactions at PKC putative consensus sites can render α4β2 nAChRs almost insensitive to nicotine without substantial effects on normal AChR function. Therefore, these PKC consensus sites in the M3–M4 cytoplasmic loop of the α4 nAChR subunit could be a target for smoking cessation drugs.

Single Aspartate Substitutions at Multiple Deep-Pore Positions Lead to Constitutively Open BK Channels

The pore-lining amino acids of ion channel proteins reside on the interface between a polar (the pore) and a non-polar environment (the rest of the protein). Manipulating the charge states of their side-chains, by tilting the energy balance between a buried and a pore-exposed conformation, can reveal important details about gating-related structural dynamics. For example, charged side-chains at the M314 position in the BK channel stabilize an open conformation. The M314D mutant, in particular, led to constitutive channel activities. We have now scanned the S6 region (I308 to N328) with aspartate replacement. We found 3 other positions where aspartate substitutions led to constitutively open BK channels in nominal zero calcium and neutral pH. These mutants are L312D, A313D and A316D. Together with M314, these positions are all located within the deep-pore region of the channel, which is immediately intracellular to the selectivity filter but more extracellular to the lower-pore region that aligns with the Kv channel “bundling crossing”. These findings provide further evidence that the BK channel pore is different from that of Kv channels, where aspartate substitution at a single position (as opposed to multiple positions in BK) in the lower-pore region (as opposed to the deep-pore region in BK) led to constitutive channel activities. The aspartate mediated constitutive activities in Kv channels is believed to result from the disruption of specific hydrophobic interactions in the “bundle-crossing” gate of a closed channel. Our observations with BK channels are unlikely to result from disruption of specific interactions, as multiple positions in the BK deep-pore, some of them adjacent along the S6 helix, showed aspartate mediated constitutive activities.

PTP-1B Is the Protein Tyrosine Phosphatase That Initiates Integrin αIIbβ3 Outside-In Signaling in Platelets.

Outside-in signaling is triggered by αIIbβ3 interaction with fibrinogen or von Willebrand factor and is required for effective platelet thrombus formation on damaged vascular surfaces. It requires ligand-induced αIIbβ3 clustering, which results in the activation of several Src family tyrosine kinases that are directly associated with αIIbβ3. However, the mechanism of Src activation by αIIbβ3 is incompletely understood. Here we demonstrate that the protein tyrosine phosphatase, PTP-1B, is required for integrin activation of c-Src and for outside-in signaling in platelets. In human or mouse platelets, c-Src was constitutively-associated with αIIbβ3 through an interaction that involved the β3 cytoplasmic domain and the c-Src SH3 domain. In resting platelets, the catalytic activity of this pool of c-Src was relatively low due to auto-inhibitory constraints imposed, in part, by an intramolecular interaction between the SH2 domain and the C-terminus of c-Src. This was promoted by phosphorylation of the C-terminal tyrosine of c-Src, tyrosine 529, by the Csk kinase. When platelets became adherent to fibrinogen or bound soluble fibrinogen in response to MnCl2, PTP-1B became associated with αIIbβ3 and c-Src, as determined by co-immunoprecipitation. At the same time, Csk dissociated from the integrin complex, c-Src tyrosine 529 became dephosphorylated, and c-Src became activated, as measured by phosphorylation of activation loop tyrosine 418. In marked contrast, gene-targeted mouse platelets deficient in PTP-1B (PTP-1B−/−) exhibited no dephosphorylation of c-Src tyrosine 529 and no activation of c-Src in response to fibrinogen binding. Furthermore, unlike wild-type platelets, PTP-1B−/− platelets exhibited little or no fibrinogen-dependent tyrosine phosphorylation of c-Src substrates, and they failed to undergo cytoskeletal reorganization or to spread on fibrinogen. These abnormalities of PTP-1B−/− platelets were due to defective outside-in signaling because surface expression of αIIbβ3 was normal, and fibrinogen bound normally in response to platelet stimulation with ADP or PAR-4 receptor agonists. When Fura-2-loaded platelets were infused and visualized by real time intravital microscopy during arterial thrombus development following laser injury to the cremaster microcirculation of a living mouse, PTP-1B−/− platelets exhibited significantly reduced intracellular free Ca2+ responses and formed smaller, less stable thrombi compared to wild-type platelets. Altogether, these studies establish that PTP-1B is a necessary, positive regulator of αIIbβ3-associated c-Src during the initiation phase of outside-in signaling in platelets. Consequently, disruption of specific interactions between αIIbβ3, c-Src and PTP-1B, or abnormalities in the activation of integrin-associated c-Src or PTP-1B, may cause defects in primary hemostasis. Moreover, these interactions provide potential targets for pharmacological blockade of outside-in signaling during arterial thrombosis.