ABSTRACT
Store-operated Ca(2+) entry (SOCE) channels are the main pathway of Ca(2+) entry in non-excitable cells such as neural progenitor cells (NPCs). However, the role of SOCE channels has not been defined in the neuronal differentiation from NPCs. Here, we show that canonical transient receptor potential channel (TRPC) as SOCE channel influences the induction of the neuronal differentiation of A2B5(+) NPCs isolated from postnatal-12-day rat cerebrums. The amplitudes of SOCE were significantly higher in neural cells differentiated from proliferating A2B5(+) NPCs and applications of SOCE blockers, 2-aminoethoxy-diphenylborane (2-APB), and ruthenium red (RR), inhibited their rise of SOCE. Among TRPC subtypes (TRPC1-7), marked expression of TRPC5 and TRPC6 with turned-off TRPC1 expression was observed in neuronal cells differentiated from proliferating A2B5(+) NPCs. TRPC5 small interfering RNA (siRNA) blocked the neuronal differentiation from A2B5(+) NPCs and reduced the rise of SOCE. In contrast, TRPC6 siRNA had no significant effect on the neuronal differentiation from A2B5(+) NPCs. These results indicate that calcium regulation by TRPC5 would play a key role as a switch between proliferation and neuronal differentiation from NPCs.
Subject(s)
Cell Differentiation , Neurons/cytology , Stem Cells/cytology , Stem Cells/metabolism , TRPC Cation Channels/metabolism , Animals , Boron Compounds/pharmacology , Calcium Signaling/drug effects , Cell Differentiation/drug effects , Cell Proliferation/drug effects , Cell Separation , Cell Survival/drug effects , Cells, Cultured , Gene Knockdown Techniques , Neuroglia/cytology , Neuroglia/drug effects , Neuroglia/metabolism , Neurons/drug effects , Neurons/metabolism , RNA, Small Interfering/metabolism , Rats , Rats, Sprague-Dawley , Stem Cells/drug effects , TRPC Cation Channels/genetics , Thapsigargin/pharmacologyABSTRACT
Ion channels are commonly expressed in recombinant forms with peptide tags, which facilitates their molecular and electrophysiological studies. However, peptide tags may alter ion channel properties. Here we describe the differential effect of peptide tags on the biochemical properties of transient receptor potential vanilloid 6 (TRPV6) channels. Yellow fluorescent protein (YFP)-tagged wild-type TRPV6 (YFP-TRPV6(WT)) showed much lower levels of aggregate-like bands in Western blots than those of Myc-TRPV6(WT). By contrast, the glycosylation level was higher with Myc-TRPV6(WT) than that with YFP-TRPV6(WT). We additionally demonstrate that peptide tags affect the protein integrity of TRPV6 channels. Myc-TRPV6(WT) was expressed as an intact channel, whereas the pore mutants Myc-TRPV6(D542A) and Myc-TRPV6(D542K) were observed to be partially fragmented. By contrast, all YFP-tagged channels were intact, although the YFP-tagged pore mutants were less glycosylated than YFP-TRPV6(WT). However, regardless of the peptide tag used, TRPV6(D542A) and TRPV6(D542K) electrophysiologically inhibited TRPV6(WT) which indicates that all pore mutants are equivalent electrophysiologically, not biochemically. Thus, our findings suggest that peptide tags can produce unintended biochemical changes of ion channels which highlight the necessity of careful biochemical evaluation to clarify the roles of ion channels.
Subject(s)
Calcium Channels/chemistry , Peptides/metabolism , TRPV Cation Channels/chemistry , Blotting, Western , Calcium/metabolism , Calcium Channels/genetics , Calcium Channels/metabolism , Cell Line , Epitopes/chemistry , Genes, myc , Glycosylation , Humans , Ligands , Luminescent Proteins/chemistry , Mutation , Plasmids , Proteins/metabolism , TRPV Cation Channels/genetics , TRPV Cation Channels/metabolism , TransfectionABSTRACT
Agonist-induced internalization of metabotropic glutamate receptors (mGluRs) plays an important role in neuronal signaling. Although internalization of mGluRs has been reported to be mediated by clathrin-dependent pathway, studies describing clathrin-independent pathways are emerging. Here, we report that agonist-induced internalization of mGluR1alpha is mediated by caveolin. We show that two caveolin-binding motifs of mGluR1alpha interact with caveolin1/2. Using cell surface-immunoprecipitation and total internal reflection fluorescence imaging, we found that agonist-induced internalization of mGluR1alpha is regulated by caveolin-binding motifs of the receptor in heterologous cells. Moreover, in the cerebellum, group I mGluR agonist dihydroxyphenylglycol increased the interaction of phosphorylated caveolin with mGluR1alpha. This interaction was blocked by methyl-beta-cyclodextrin, known to disrupt caveolin/caveolae-dependent signaling by cholesterol depletion. Methyl-beta-cyclodextrin also blocked the agonist-induced internalization of mGluR1alpha. Thus, these findings represent the evidence for agonist-induced internalization of mGluR1alpha via caveolin and suggest that caveolin might play a role in synaptic metaplasticity by regulating internalization of mGluR1alpha in the cerebellum.