The activation mechanism of the classical transient receptor potential channels TRPC4 and -5 via the G q/11 protein-phospholipase C (PLC) signaling pathway has remained elusive so far. In contrast to all other TRPC channels, the PLC product diacylglycerol (DAG) is not sufficient for channel activation, whereas TRPC4/5 channel activity is potentiated by phosphatidylinositol 4,5-bisphosphate (PIP 2 ) depletion. As a characteristic structural feature, TRPC4/5 channels contain a C-terminal PDZ-binding motif allowing for binding of the scaffolding proteins Na+ exchanger regulatory factor (NHERF) 1 and 2. PKC inhibition or the exchange of threonine for alanine in the C-terminal PDZ-binding motif conferred DAG sensitivity to the channel. Altogether, we present a DAG-mediated activation mechanism for TRPC4/5 channels tightly regulated by NHERF1/2 interaction. PIP 2 depletion evokes a C-terminal conformational change of TRPC5 proteins leading to dynamic dissociation of NHERF1/2 from the C terminus of TRPC5 as a prerequisite for DAG sensitivity. We show that NHERF proteins are direct regulators of ion channel activity and that DAG sensitivity is a distinctive hallmark of TRPC channels.and -5 channels are members of the classical transient receptor potential cation (TRPC) family of nonselective, calcium permeable receptor-operated cation channels. They are widely expressed in many tissues, including brain, kidney, and the vascular system. High expression levels are found in the central nervous system where TRPC4 and -5 are involved in amygdala function and fear-related behavior (1, 2), seizure, and excitotoxicity (3). Furthermore, TRPC5 channels are implicated in neuronal depolarization and bursting during epiletiform seizures (4) and regulate hippocampal neurite length and growth cone morphology (5). In the kidney, TRPC5 channels are proposed to be protective against renal failure (6). TRPC channels are usually activated by G q/11 proteincoupled receptors via phospholipase C (PLC) activation resulting in cleavage of phosphatidylinositol-3,4-bisphosphate (PIP 2 ) into the second messengers inositol-1,4,5-trisphosphate (IP 3 ) and diacylglycerol (DAG). DAG is known to activate TRPC2, -3, -6, and -7 (7-9) channels, whereas TRPC4 and -5 are supposed to be insensitive to the PLC product DAG (8) and are even inhibited by DAG or its membrane-permeable analog 1-oleoyl-2-acetyl-sn-glycerol (OAG) (10). DAG-mediated TRPC5 channel inhibition was shown to be PKC dependent (10). Furthermore, TRPC4 and -5 channels can be activated by depleting PIP 2 (11, 12), contrary to TRPC6 and -7 channels, which are inhibited by PIP 2 depletion (13). However, there are first hints to show that endogenously expressed TRPC5 channels might be DAG sensitive (14) but mechanistic insight is lacking so far.A noteworthy structural difference between TRPC4 and -5 and the established DAG-sensitive TRPC3, -6, and -7 channels is the PDZbinding motif VTTLR in the C termini of TRPC4 and -5 channels (15-17) as a structural basis of the interaction with Na + /H + e...

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Although there are a large number of studies available for the evaluation of the therapeutic efficacy of targeted polymeric nanoparticles, little is known about the critical attributes that can further influence their uptake into target cells. In this study, varying cRGD ligand densities (0− 100% surface functionalization) were combined with different poly(ethylene glycol) (PEG) spacer lengths (2/3.5/5 kDa), and the specific receptor binding of targeted core−shell structured poly(lactic-co-glycolic acid)/poly(lactic acid)−PEG nanoparticles was evaluated using α v β 3 integrin-overexpressing U87MG glioblastoma cells. Nanoparticles with 100% surface functionalization and short PEG2k linkers displayed a high propensity to form colloidal clusters, allowing for the cooperative binding to integrin receptors on the cellular membrane. In contrast, the high flexibility of longer PEG chains enhanced the chance of ligand entanglement and shrouding, decreasing the number of ligand−receptor binding events. As a result, the combination of short PEG2k linkers and a high cRGD surface modification synergistically increased the uptake of nanoparticles into target cells. Even though to date, the nanoparticle size and its degree of functionalization are considered to be the major determinants for controlling the uptake efficiency of targeted colloids, these results strongly suggest that the role of the linker length should be carefully taken into consideration for the design of targeted drug delivery formulations to maximize the therapeutic efficacy and minimize adverse side effects.

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Formation of a protein corona can severely compromise the performance of functionalized polymeric nanoparticles by changing their identity and integrity.