The Rho family of small guanosine triphosphatases (Rho GTPases: RhoA, Cdc42, and Rac1) regulates many aspects of cell behavior, including actin dynamics and cell migration. The generation of calcium ion (Ca2+) microdomains is critical in promoting cell migration because they control the localized activity of Rho GTPases. We identified receptor-activated TRPC5 and TRPC6 (transient receptor potential canonical type 5 and 6) channels as antagonistic regulators of actin remodeling and cell motility in fibroblasts and kidney podocytes. We show that TRPC5 is in a molecular complex with Rac1, whereas TRPC6 is in a molecular complex with RhoA. TRPC5-mediated Ca2+ influx induces Rac1 activation, thereby promoting cell migration, whereas TRPC6-mediated Ca2+ influx increases RhoA activity, thereby inhibiting cell migration. Our data unveil antagonistic Ca2+ influx pathways as a conserved signaling mechanism for the integrated regulation of cell migration.

The genome of the cnidarian Nematostella vectensis (starlet sea anemone) provides a molecular genetic view into the first nervous systems, which appeared in a late common ancestor of cnidarians and bilaterians. Nematostella has a surprisingly large and diverse set of neuronal signaling genes including paralogs of most neuronal signaling molecules found in higher metazoans. Several ion channel gene families are highly expanded in the sea anemone, including three subfamilies of the Shaker K+ channel gene family: Shaker (Kv1), Shaw (Kv3) and Shal (Kv4). In order to better understand the physiological significance of these voltage-gated K+ channel expansions, we analyzed the function of 18 members of the 20 gene Shaker subfamily in Nematostella. Six of the Nematostella Shaker genes express functional homotetrameric K+ channels in vitro. These include functional orthologs of bilaterian Shakers and channels with an unusually high threshold for voltage activation. We identified 11 Nematostella Shaker genes with a distinct “silent” or “regulatory” phenotype; these encode subunits that function only in heteromeric channels and serve to further diversify Nematostella Shaker channel gating properties. Subunits with the regulatory phenotype have not previously been found in the Shaker subfamily, but have evolved independently in the Shab (Kv2) family in vertebrates and the Shal family in a cnidarian. Phylogenetic analysis indicates that regulatory subunits were present in ancestral cnidarians, but have continued to diversity at a high rate after the split between anthozoans and hydrozoans. Comparison of Shaker family gene complements from diverse metazoan species reveals frequent, large scale duplication has produced highly unique sets of Shaker channels in the major metazoan lineages.

Transmembrane mucins are highly -glycosylated glycoproteins that coat the apical glycocalyx on mucosal surfaces and represent the first line of cellular defense against infection and injury. Relatively low levels of-glycans are found on transmembrane mucins, and their structure and function remain poorly characterized. We previously reported that carbohydrate-dependent interactions of transmembrane mucins with galectin-3 contribute to maintenance of the epithelial barrier at the ocular surface. Now, using MALDI-TOF mass spectrometry, we report that transmembrane mucin -glycans in differentiated human corneal epithelial cells contain primarily complex-type structures with-acetyllactosamine, a preferred galectin ligand. In -glycosylation inhibition experiments, we find that treatment with tunicamycin and siRNA-mediated knockdown of the Golgi-acetylglucosaminyltransferase I gene () induce partial loss of both total and cell-surface levels of the largest mucin, MUC16, and a concomitant reduction in glycocalyx barrier function. Moreover, we identified a distinct role for -glycans in promoting MUC16's binding affinity toward galectin-3 and in causing retention of the lectin on the epithelial cell surface. Taken together, these studies define a role for-linked oligosaccharides in supporting the stability and function of transmembrane mucins on mucosal surfaces.

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Synonymous single nucleotide polymorphisms (SNPs) within a transcript's coding region produce no change in the amino acid sequence of the protein product and are therefore intuitively assumed to have a neutral effect on protein function. We report that two common variants of high-temperature requirement A1 (HTRA1) that increase the inherited risk of neovascular agerelated macular degeneration (NvAMD) harbor synonymous SNPs within exon 1 of HTRA1 that convert common codons for Ala34 and Gly36 to less frequently used codons. The frequent-to-rare codon conversion reduced the mRNA translation rate and appeared to compromise HtrA1's conformation and function. The protein product generated from the SNP-containing cDNA displayed enhanced susceptibility to proteolysis and a reduced affinity for an anti-HtrA1 antibody. The NvAMD-associated synonymous polymorphisms lie within HtrA1's putative insulin-like growth factor 1 (IGF-1) binding domain. They reduced HtrA1's abilities to associate with IGF-1 and to ameliorate IGF-1-stimulated signaling events and cellular responses. These observations highlight the relevance of synonymous codon usage to protein function and implicate homeostatic protein quality control mechanisms that may go awry in NvAMD. Single nucleotide polymorphisms (SNPs) that fall within the coding region have the potential to alter the amino acid sequence of a gene's product and therefore can serve as a Rosetta stone for understanding the pathogenesis of human disorders (1, 2). A curiosity that emerged from human genome-wide association studies is that exonic SNPs that do not alter the amino acid sequence of the protein product (i.e., SNPs that are synonymous) are as common as SNPs that do (i.e., SNPs that are nonsynonymous) (3). Furthermore, some of the disease-associated synonymous SNPs constitute the molecular underpinnings of pathology, meaning that they are enriched in affected human subjects and alter the gene product. For the majority (95%) of disease-associated synonymous SNPs, aberrant gene products were attributed to unstable mRNA transcripts with a reduced half-life or mutations in splice sites that resulted in exon skipping (4, 5). In other cases, synonymous SNPs caused translational defects independently of mRNA splicing errors (6-9).A parsimonious mechanism by which synonymous SNPs impact the integrity of protein products involves the alteration of codon usage. In vivo, folding of nascent proteins proceeds cotranslationally (10, 11) and is influenced by the rate of ribosome transit through the mRNA template. What distinguishes synonymous codons that encode a given degenerate amino acid is the abundance of their corresponding tRNA, which is lower for infrequently used codons than for frequently used codons (12, 13). Consequently, codon frequency can influence the rate of translation. Of note, codon bias has been widely described in prokaryotes and single-celled eukaryotes but less extensively in complex eukaryotes.In humans, correlations between optimum codon usage and either protein expression...

The incretin peptides, glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide-1 (GLP-1), potentiate glucose-stimulated insulin secretion (GSIS) and ␤-cell proliferation and differentiation. Ca 2ϩ influx via voltage-gated L-type Ca 2ϩ channels is required for GLP-1 and GIP potentiation of GSIS. We investigated the role of the L-type Ca stores, PKA and PKC activity, and activation of ERK1/2.

Using insulin-secreting cell line (INS)-1 cells stably expressing dihydropyridine-insensitive mutants of either Ca v 1.2 or Ca v 1.3, we previously demonstrated that Ca v 1.3 is preferentially coupled to insulin secretion and [Ca 2ϩ ] i oscillations stimulated by 11.2 mM glucose. Using the same system, we found that insulin secretion in 7.5 mM glucose plus 1 mM 8-bromo-cAMP (8-BrcAMP) is mediated by both Ca v 1.2 and Ca v 1. and ryanodine (0.5 M). In contrast, ryanodine has no effect on insulin secretion or [Ca 2ϩ ] i oscillations stimulated by 11.2 mM glucose in INS-1 cells. Our data suggest that both Ca v 1.2 and Ca v 1.3 mediate insulin secretion stimulated by 7.5 mM glucose and cAMP via a mechanism that requires internal stores of Ca 2ϩ . Furthermore, cAMP modulation of secretion mediated by Ca v 1.2 seems to involve both Epac2 and PKA independently. In contrast, cAMP modulation of Ca v 1.3-mediated secretion depends upon PKA activation, whereas the contribution of Epac2 is dependent upon PKA activation.Metabolism of glucose to ATP in pancreatic ␤-cells results in the closure of K ATP channels, which depolarizes the plasma membrane and activates voltage-dependent calcium channels (VDCCs) (Rajan et al., 1990). Calcium influx via L-type VDCCs causes an elevation of intracellular calcium concentration ([Ca 2ϩ ] i ) and triggers exocytosis of insulincontaining granules (Wollheim and Sharp, 1981). Glucagonlike peptide (GLP)-1 is an insulin secretagogue hormone secreted by intestinal L cells that can potentiate insulin secretion from ␤-cells in response to elevated blood glucose (Schmidt et al., 1985). The insulinotropic effect of GLP-1 has been widely recognized and intensively studied because of its potential therapeutic application in the treatment of noninsulin-dependent (type II) diabetes mellitus (Nauck et al., 1993a,b).The glucose dependence of the insulinotropic action of GLP-1 implies that there is cross-talk between the L-type H89,amino)ethyl]-5-isoquinolinesulfonamide, 2HCl; Ca v 1.2/DHPi, Ca v 1.2 channel insensitive to dihydropyridines; Ca v 1.3/DHPi, Ca v 1.3 channel insensitive to dihydropyridines; Rp-cAMPS, adenosine 3Ј,5Ј-cyclic monophosphorothioate (Rp-isomer); GFP, green fluorescent protein; DHPi, dihydropyridine-insensitive; KRBH, Krebs-Ringer-bicarbonate HEPES; ANOVA, analysis of variance; ER, endoplasmic reticulum; RyR, ryanodine receptor; IP 3 R, inositol 1,4,5-triphosphate receptors.

L-type Ca 2ϩ channels play a key role in the integration of physiological signals regulating insulin secretion that probably requires their localization to specific subdomains of the plasma membrane. We investigated the role of the intracellular II-III loop domains of the L-type channels Ca v 1.2 and 1.