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[Figure: see text].

Excessive glucocorticoid (GC) production in adipose tissue promotes the development of visceral obesity and metabolic syndrome (MS). 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) is critical for controlling intracellular GC production, and this process is tightly regulated by hexose-6-phosphate dehydrogenase (H6PDH). To better understand the integrated molecular physiological effects of adipose H6PDH, we created a tissue-specific knockout of the H6PDH gene mouse model in adipocytes (adipocyte-specific conditional knockout of H6PDH (H6PDHAcKO) mice). H6PDHAcKO mice exhibited almost complete absence of H6PDH expression and decreased intra-adipose corticosterone production with a reduction in 11β-HSD1 activity in adipose tissue. These mice also had decreased abdominal fat mass, which was paralleled by decreased adipose lipogenic acetyl-CoA carboxylase (ACC) and ATP-citrate lyase (ACL) gene expression and reduction in their transcription factor C/EBPα mRNA levels. Moreover, H6PDHAcKO mice also had reduced fasting blood glucose levels, increased glucose tolerance, and increased insulin sensitivity. In addition, plasma free fatty acid (FFA) levels were decreased with a concomitant decrease in the expression of lipase adipose triglyceride lipase (ATGL) and hormone-sensitive lipase (HSL) in adipose tissue. These results indicate that inactivation of adipocyte H6PDH expression is sufficient to cause intra-adipose GC inactivation that leads to a favorable pattern of metabolic phenotypes. These data suggest that H6PDHAcKO mice may provide a good model for studying the potential contributions of fat-specific H6PDH inhibition to improve the metabolic phenotype in vivo. Our study suggests that suppression or inactivation of H6PDH expression in adipocytes could be an effective intervention for treating obesity and diabetes.

Hair plays important roles, ranging from the conservation of body heat to the preservation of psychological well-being. Hair loss or alopecia affects millions worldwide, but methods that can be used to regrow hair are lacking. We report that quiescent (telogen) hair follicles can be stimulated to initiate anagen and hair growth by small molecules that activate autophagy, including the metabolites α-ketoglutarate (α-KG) and α-ketobutyrate (α-KB), and the prescription drugs rapamycin and metformin, which impinge on mTOR and AMPK signaling. Stimulation of hair growth by these agents is blocked by specific autophagy inhibitors, suggesting a mechanistic link between autophagy and hair regeneration. Consistently, increased autophagy is detected upon anagen entry during the natural hair follicle cycle, and oral α-KB prevents hair loss in aged mice. Our finding that anagen can be pharmacologically activated in telogen skin when natural anagen-inducing signal(s) are absent has implications for the treatment of hair loss patients.

BackgroundVisceral fat accumulation increases the risk of developing type 2 diabetes and metabolic syndrome, and is associated with excessive glucocorticoids (GCs). Fat depot-specific GC action is tightly controlled by 11ß-hydroxysteroid dehydrogenase (11ß-HSD1) coupled with the enzyme hexose-6-phosphate dehydrogenase (H6PDH). Mice with inactivation or activation of H6PDH genes show altered adipose 11ß-HSD1 activity and lipid storage. We hypothesized that adipose tissue H6PDH activation is a leading cause for the visceral obesity and insulin resistance. Here, we explored the role and possible mechanism of enhancing adipose H6PDH in the development of visceral adiposity in vivo.MethodsWe investigated the potential contribution of adipose H6PDH activation to the accumulation of visceral fat by characterization of visceral fat obese gene expression profiles, fat distribution, adipocyte metabolic molecules, and abdominal fat-specific GC signaling mechanisms underlying the diet-induced visceral obesity and insulin resistance in H6PDH transgenic mice fed a standard of high-fat diet (HFD).ResultsTransgenic H6PDH mice display increased abdominal fat accumulation, which is paralleled by elevated lipid synthesis associated with induction of lipogenic transcriptor C/EBPα and PPARγ mRNA levels within adipose tissue. Transgenic H6PDH mice fed a high-fat diet (HFD) gained more abdominal visceral fat mass coupled with activation of GSK3β and induction of XBP1/IRE1α, but reduced pThr308 Akt/PKB content and browning gene CD137 and GLUT4 mRNA levels within the visceral adipose tissue than WT controls. HFD-fed H6PDH transgenic mice also had impaired insulin sensitivity and exhibited elevated levels of intra-adipose GCs with induction of adipose 11ß-HSD1.ConclusionThese data provide the first in vivo mechanistic evidence for the adverse metabolic effects of adipose H6PDH activation on visceral fat distribution, fat metabolism, and adipocyte function through enhancing 11ß-HSD1-driven intra-adipose GC action.

More than 60 members of the Rab family of guanosine triphosphatases (GTPases) exist in the human genome. Rab GTPases are small proteins that are primarily involved in the formation, trafficking, and fusion of vesicles. We showed thatCRACR2A(Ca(2+) release-activated Ca(2+) channel regulator 2A) encodes a lymphocyte-specific large Rab GTPase that contains multiple functional domains, including EF-hand motifs, a proline-rich domain (PRD), and a Rab GTPase domain with an unconventional prenylation site. Through experiments involving gene silencing in cells and knockout mice, we demonstrated a role for CRACR2A in the activation of the Ca(2+) and c-Jun N-terminal kinase signaling pathways in response to T cell receptor (TCR) stimulation. Vesicles containing this Rab GTPase translocated from near the Golgi to the immunological synapse formed between a T cell and a cognate antigen-presenting cell to activate these signaling pathways. The interaction between the PRD of CRACR2A and the guanidine nucleotide exchange factor Vav1 was required for the accumulation of these vesicles at the immunological synapse. Furthermore, we demonstrated that GTP binding and prenylation of CRACR2A were associated with its localization near the Golgi and its stability. Our findings reveal a previously uncharacterized function of a large Rab GTPase and vesicles near the Golgi in TCR signaling. Other GTPases with similar domain architectures may have similar functions in T cells.

We discovered recently that the central metabolite α-ketoglutarate (α-KG) extends the lifespan of C. elegans through inhibition of ATP synthase and TOR signaling. Here we find, unexpectedly, that (R)-2-hydroxyglutarate ((R)-2HG), an oncometabolite that interferes with various α-KG-mediated processes, similarly extends worm lifespan. (R)-2HG accumulates in human cancers carrying neomorphic mutations in the isocitrate dehydrogenase (IDH) 1 and 2 genes. We show that, like α-KG, both (R)-2HG and (S)-2HG bind and inhibit ATP synthase and inhibit mTOR signaling. These effects are mirrored in IDH1 mutant cells, suggesting a growth-suppressive function of (R)-2HG. Consistently, inhibition of ATP synthase by 2-HG or α-KG in glioblastoma cells is sufficient for growth arrest and tumor cell killing under conditions of glucose limitation, e.g., when ketone bodies (instead of glucose) are supplied for energy. These findings inform therapeutic strategies and open avenues for investigating the roles of 2-HG and metabolites in biology and disease.

Long-term glucocorticoid exposure increases the risk for developing type 2 diabetes. Prereceptor activation of glucocorticoid availability in target tissue by 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) coupled with hexose-6-phosphate dehydrogenase (H6PDH) is an important mediator of the metabolic syndrome. We explored whether the tissue-specific modulation of 11β-HSD1 and H6PDH in adipose tissue mediates glucocorticoid-induced insulin resistance and lipolysis and analyzed the effects of 11β-HSD1 inhibition on the key lipid metabolism genes and insulin-signaling cascade. We observed that corticosterone (CORT) treatment increased expression of 11β-HSD1 and H6PDH and induced lipase HSL and ATGL with suppression of p-Thr(172) AMPK in adipose tissue of C57BL/6J mice. In contrast, CORT induced adipose insulin resistance, as reflected by a marked decrease in IR and IRS-1 gene expression with a reduction in p-Thr(308) Akt/PKB. Furthermore, 11β-HSD1 shRNA attenuated CORT-induced 11β-HSD1 and lipase expression and improved insulin sensitivity with a concomitant stimulation of pThr(308) Akt/PKB and p-Thr(172) AMPK within adipose tissue. Addition of CORT to 3T3-L1 adipocytes enhanced 11β-HSD1 and H6PDH and impaired p-Thr(308) Akt/PKB, leading to lipolysis. Knockdown of 11β-HSD1 by shRNA attenuated CORT-induced lipolysis and reversed CORT-mediated inhibition of pThr(172) AMPK, which was accompanied by a parallel improvement of insulin signaling response in these cells. These findings suggest that elevated adipose 11β-HSD1 expression may contribute to glucocorticoid-induced insulin resistance and adipolysis.

PurposeThe clinical management of cataracts in infancy involves surgical removal of the lens to ensure transmission of light to the retina, which is essential for normal neural development of the infant. This surgery, however, entails a lifelong follow-up and impaired vision. To our knowledge, no animal models recapitulate human lamellar opacities, the most prevalent form of early childhood cataracts. We present data on the recreation of the human lamellar cataract phenotype in transgenic mice.MethodsMutations in the DNA binding domain (DBD) of the heat shock transcription factor 4 (HSF4) are known to be associated with early childhood autosomal dominant lamellar cataract. We used bacterial artificial chromosome (BAC) transgenesis to express a hybrid gene: Hsf4 (DBD)-enhanced green fluorescent protein (EGFP), by recombineering EGFP sequences into the DBD of the Hsf4 gene, to interfere with the DNA binding properties of Hsf4.ResultsWe recapitulated the human lamellar cataract, in its temporal as well as spatial presentation, within the transgenic mouse lens. This phenotype was reproduced faithfully using four different BACs, indicating that EGFP can be used to target transcription factor function in transgenic mice. Molecular and cell biological examination of early postnatal transgenic lens reveals impairment of secondary fiber cell differentiation.ConclusionsRecreation of the human lamellar cataract phenotype in mice allows investigation of this human pathology at a level not possible previously and points to the relevance of fiber cell heterogeneity dictated by fiber cell-specific gene activity in the biogenesis of the lamellar cataract.

Metabolism and ageing are intimately linked. Compared with ad libitum feeding, dietary restriction consistently extends lifespan and delays age-related diseases in evolutionarily diverse organisms. Similar conditions of nutrient limitation and genetic or pharmacological perturbations of nutrient or energy metabolism also have longevity benefits. Recently, several metabolites have been identified that modulate ageing; however, the molecular mechanisms underlying this are largely undefined. Here we show that α-ketoglutarate (α-KG), a tricarboxylic acid cycle intermediate, extends the lifespan of adult Caenorhabditis elegans. ATP synthase subunit β is identified as a novel binding protein of α-KG using a small-molecule target identification strategy termed drug affinity responsive target stability (DARTS). The ATP synthase, also known as complex V of the mitochondrial electron transport chain, is the main cellular energy-generating machinery and is highly conserved throughout evolution. Although complete loss of mitochondrial function is detrimental, partial suppression of the electron transport chain has been shown to extend C. elegans lifespan. We show that α-KG inhibits ATP synthase and, similar to ATP synthase knockdown, inhibition by α-KG leads to reduced ATP content, decreased oxygen consumption, and increased autophagy in both C. elegans and mammalian cells. We provide evidence that the lifespan increase by α-KG requires ATP synthase subunit β and is dependent on target of rapamycin (TOR) downstream. Endogenous α-KG levels are increased on starvation and α-KG does not extend the lifespan of dietary-restricted animals, indicating that α-KG is a key metabolite that mediates longevity by dietary restriction. Our analyses uncover new molecular links between a common metabolite, a universal cellular energy generator and dietary restriction in the regulation of organismal lifespan, thus suggesting new strategies for the prevention and treatment of ageing and age-related diseases.

Metabolism and ageing are intimately linked. Compared with ad libitum feeding, dietary restriction consistently extends lifespan and delays age-related diseases in evolutionarily diverse organisms. Similar conditions of nutrient limitation and genetic or pharmacological perturbations of nutrient or energy metabolism also have longevity benefits. Recently, several metabolites have been identified that modulate ageing; however, the molecular mechanisms underlying this are largely undefined. Here we show that α-ketoglutarate (α-KG), a tricarboxylic acid cycle intermediate, extends the lifespan of adult Caenorhabditis elegans. ATP synthase subunit β is identified as a novel binding protein of α-KG using a small-molecule target identification strategy termed drug affinity responsive target stability (DARTS). The ATP synthase, also known as complex V of the mitochondrial electron transport chain, is the main cellular energy-generating machinery and is highly conserved throughout evolution. Although complete loss of mitochondrial function is detrimental, partial suppression of the electron transport chain has been shown to extend C. elegans lifespan. We show that α-KG inhibits ATP synthase and, similar to ATP synthase knockdown, inhibition by α-KG leads to reduced ATP content, decreased oxygen consumption, and increased autophagy in both C. elegans and mammalian cells. We provide evidence that the lifespan increase by α-KG requires ATP synthase subunit β and is dependent on target of rapamycin (TOR) downstream. Endogenous α-KG levels are increased on starvation and α-KG does not extend the lifespan of dietary-restricted animals, indicating that α-KG is a key metabolite that mediates longevity by dietary restriction. Our analyses uncover new molecular links between a common metabolite, a universal cellular energy generator and dietary restriction in the regulation of organismal lifespan, thus suggesting new strategies for the prevention and treatment of ageing and age-related diseases.

BACKGROUND: In a classic model, G(i)α proteins including G(i1)α, G(i2)α and G(i3)α are important for transducing signals from G(i)α protein-coupled receptors (G(i)αPCRs) to their downstream cascades in response to hormones and neurotransmitters. Our previous study has suggested that G(i1)α, G(i2)α and G(i3)α are also important for the activation of the PI3K/Akt/mTORC1 pathway by epidermal growth factor (EGF) and its family members. However, a genetic role of these G(i)α proteins in the activation of extracellular signal-regulated protein kinase 1 and 2 (ERK1/2) by EGF is largely unknown. Further, it is not clear whether these G(i)α proteins are also engaged in the activation of both the Akt/mTORC1 and ERK1/2 pathways by other growth factor family members. Additionally, a role of these G(i)α proteins in breast cancer remains to be elucidated. RESULTS: We found that Gi1/3 deficient MEFs with the low expression level of G(i2)α showed defective ERK1/2 activation by EGFs, IGF-1 and insulin, and Akt and mTORC1 activation by EGFs and FGFs. Gi1/2/3 knockdown breast cancer cells exhibited a similar defect in the activations and a defect in in vitro growth and invasion. The G(i)α proteins associated with RTKs, Gab1, FRS2 and Shp2 in breast cancer cells and their ablation impaired Gab1s interactions with Shp2 in response to EGF and IGF-1, or with FRS2 and Grb2 in response to bFGF. CONCLUSIONS: G(i)α proteins differentially regulate the activation of Akt, mTORC1 and ERK1/2 by different families of growth factors. G(i)α proteins are important for breast cancer cell growth and invasion.

Abstract Background Reciprocal interactions between lung mesenchymal and epithelial cells play essential roles in lung organogenesis and homeostasis. Although the molecular markers and related animal models that target lung epithelial cells are relatively well studied, molecular markers of lung mesenchymal cells and the genetic tools to target and/or manipulate gene expression in a lung mesenchyme-specific manner are not available, which becomes a critical barrier to the study of lung mesenchymal biology and the related pulmonary diseases. Results We have identified a mouse Tbx4 gene enhancer that contains conserved DNA sequences across many vertebrate species with lung or lung-like gas exchange organ. We then generate a mouse line to express rtTA/LacZ under the control of the Tbx4 lung enhancer, and therefore a Tet-On inducible transgenic system to target lung mesenchymal cells at different developmental stages. By combining a Tbx4-rtTA driven Tet-On inducible Cre expression mouse line with a Cre reporter mouse line, the spatial-temporal patterns of Tbx4 lung enhancer targeted lung mesenchymal cells were defined. Pulmonary endothelial cells and vascular smooth muscle cells were targeted by the Tbx4-rtTA driver line prior to E11.5 and E15.5, respectively, while other subtypes of lung mesenchymal cells including airway smooth muscle cells, fibroblasts, pericytes could be targeted during the entire developmental stage. Conclusions Developmental lung mesenchymal cells can be specifically marked by Tbx4 lung enhancer activity. With our newly created Tbx4 lung enhancer-driven Tet-On inducible system, lung mesenchymal cells can be specifically and differentially targeted in vivo for the first time by controlling the doxycycline induction time window. This novel system provides a unique tool to study lung mesenchymal cell lineages and gene functions in lung mesenchymal development, injury repair, and regeneration in mice.

PurposeOcular Albinism type 1 (OA1) is a disease caused by mutations in the OA1 gene and characterized by the presence of macromelanosomes in the retinal pigment epithelium (RPE) as well as abnormal crossing of the optic axons at the optic chiasm. We showed in our previous studies in mice that Oa1 activates specifically Gαi3 in its signaling pathway and thus, hypothesized that a constitutively active Gαi3 in the RPE of Oa1-/- mice might keep on the Oa1 signaling cascade and prevent the formation of macromelanosomes. To test this hypothesis, we have generated transgenic mice that carry the constitutively active Gαi3 (Q204L) protein in the RPE of Oa1-/- mice and are now reporting the effects that the transgene produced on the Oa1-/- RPE phenotype.MethodsTransgenic mice carrying RPE-specific expression of the constitutively active Gαi3 (Q204L) were generated by injecting fertilized eggs of Oa1-/- females with a lentivirus containing the Gαi3 (Q204L) cDNA. PCR, Southern blots, Western blots and confocal microscopy were used to confirm the presence of the transgene in the RPE of positive transgenic mice. Morphometrical analyses were performed using electron microscopy to compare the size and number of melanosomes per RPE area in putative Oa1-/-, Gαi3 (Q204L) transgenic mice with those of wild-type NCrl and Oa1-/- mice.ResultsWe found a correlation between the presence of the constitutively active Gαi3 (Q204L) transgene and the rescue of the normal phenotype of RPE melanosomes in Oa1-/-, Gαi3 (Q204L) mice. These mice have higher density of melanosomes per RPE area and a larger number of small melanosomes than Oa1-/- mice, and their RPE phenotype is similar to that of wild-type mice.ConclusionsOur results show that a constitutively active Gαi3 protein can by-pass the lack of Oa1 protein in Oa1-/- mice and consequently rescue the RPE melanosomal phenotype.

BackgroundOcular albinism type 1, an X-linked disease characterized by the presence of enlarged melanosomes in the retinal pigment epithelium (RPE) and abnormal crossing of axons at the optic chiasm, is caused by mutations in the OA1 gene. The protein product of this gene is a G-protein-coupled receptor (GPCR) localized in RPE melanosomes. The Oa1-/- mouse model of ocular albinism reproduces the human disease. Oa1 has been shown to immunoprecipitate with the Gαi subunit of heterotrimeric G proteins from human skin melanocytes. However, the Gαi subfamily has three highly homologous members, Gαi1, Gαi2 and Gαi3 and it is possible that one or more of them partners with Oa1. We had previously shown by in-vivo studies that Gαi3-/- and Oa1-/- mice have similar RPE phenotype and decussation patterns. In this paper we analyze the specificity of the Oa1-Gαi interaction.MethodologyBy using the genetic mouse models Gαi1-/-, Gαi2-/-, Gαi3-/- and the double knockout Gαi1-/-, Gαi3-/- that lack functional Gαi1, Gαi2, Gαi3, or both Gαi1 and Gαi3 proteins, respectively, we show that Gαi3 is critical for the maintenance of a normal melanosomal phenotype and that its absence is associated with changes in melanosomal size and density. GST-pull-down and immunoprecipitation assays conclusively demonstrate that Gαi3 is the only Gαi that binds to Oa1. Western blots show that Gαi3 expression is barely detectable in the Oa1-/- RPE, strongly supporting a previously unsuspected role for Gαi3 in melanosomal biogenesis.ConclusionOur results identify the Oa1 transducer Gαi3 as the first downstream component in the Oa1 signaling pathway.

Integrins link the extracellular matrix to the cellular cytoskeleton and serve important roles in cell growth, differentiation, migration, and survival. Ablation of beta1 integrin in all murine tissues results in peri-implantation embryonic lethality. To investigate the role of beta1 integrin in the myocardium, we used Cre-LoxP technology to inactivate the beta1 integrin gene exclusively in ventricular cardiac myocytes. Animals with homozygous ventricular myocyte beta1 integrin gene excision were born in appropriate numbers and grew into adulthood. These animals had 18% of control levels of beta1D integrin protein in the heart and displayed myocardial fibrosis. High-fidelity micromanometer-tipped catheterization of the intact 5-week-old beta1 integrin knockout mice showed depressed left ventricular basal and dobutamine-stimulated contractility and relaxation (LV dP/dt(max) and LV dP/dt(min)) as compared with control groups (n=8 to 10 of each, P

Capacitative [Ca.sup.2+] entry (CCE) is [Ca.sup.2+] entering after stimulation of inositol 1,4,5-trisphosphate (IP3) formation and initiation of [Ca.sup.2+] store depletion. One hallmark of CCE is that it can also be triggered merely by store depletion, as occurs after inhibition of internal [Ca.sup.2+] pumps with thapsigargin. Evidence has accumulated in support of a role of transient receptor potential (Trp) proteins as structural subunits of a class of [Ca.sup.2+]-permeable cation channels activated by agonists that stimulate IP3 formation - very likely through a direct interaction between the IP3 receptor and a Trp subunit of the [Ca.sup.2+] entry channel. The role of Trp's in [Ca.sup.2+] entry triggered by store depletion alone is less clear. Only a few of the cloned Trp's appear to enhance this type of [Ca.sup.2+] entry, and when they do, the effect requires special conditions to be observed, which native CCE does not. Here we report the full-length cDNA of mouse trp2, the homologue of the human trp2 pseudogene. Mouse Trp2 is shown to be readily activated not only after stimulation with an agonist but also by store depletion in the absence of an agonist. In contrast to other Trp proteins, Trp2-mediated [Ca.sup.2+] entry activated by store depletion is seen under the same conditions that reveal endogenous store depletion-activated [Ca.sup.2+] entry, i.e., classical CCE. The findings support the general hypothesis that Trp proteins are subunits of store- and receptor-operated [Ca.sup.2+] channels.

Mammalian genes related to the Drosophila trp gene encode proteins that form the subunits of the channels required for agonist-activated capacitative Ca2+ entry (CCE) into mammalian cells. In COS cells, the expression of two full-length cDNA's encoding the human trp homologs, Htrp1 and Htrp3, increase CCE by 75% and 230%, respectively. Ca2+ influx due to endogenous COS cell CCE channels is more sensitive to suppression by La3+ and Ni2+ than that mediated by Htrp3. The expression of the genes in the antisense orientation inhibits CCE.

Experimental studies of the primary structure and ligand-binding behavior of a human opsin-related gene encoding a retinaldehyde binding protein from bovine retinal pigment epithelium (RPE) reveal that RPE-retinal G protein coupled receptor (RGR) binds to 11-cis- and all-trans-retinal and the amino acid sequence of the protein is 86% similar to bovine RGR sequence. The 14.8 kb gene is present as seven exons with a structure different from that of visual pigment genes. The all-trans-retinal binding property of RGR indicates that it catalyzes chromophore isomerization.

Abstract Background Transgenic animals have become valuable tools for both research and applied purposes. The current method of gene transfer, microinjection, which is widely used in transgenic mouse production, has only had limited success in producing transgenic animals of larger or higher species. Here, we report a linker based sperm-mediated gene transfer method (LB-SMGT) that greatly improves the production efficiency of large transgenic animals. Results The linker protein, a monoclonal antibody (mAb C), is reactive to a surface antigen on sperm of all tested species including pig, mouse, chicken, cow, goat, sheep, and human. mAb C is a basic protein that binds to DNA through ionic interaction allowing exogenous DNA to be linked specifically to sperm. After fertilization of the egg, the DNA is shown to be successfully integrated into the genome of viable pig and mouse offspring with germ-line transfer to the F1 generation at a highly efficient rate: 37.5% of pigs and 33% of mice. The integration is demonstrated again by FISH analysis and F2 transmission in pigs. Furthermore, expression of the transgene is demonstrated in 61% (35/57) of transgenic pigs (F0 generation). Conclusions Our data suggests that LB-SMGT could be used to generate transgenic animals efficiently in many different species.

Lack of muscarinic regulation of [Ca.sup.2+] channels in [G.sub.i2][Alpha] gene knockout mouse hearts. Am J Physiol Heart Circ Physiol 280: H1989-H1995, 2001.--The purpose of the present study was to examine the role of [G.sub.i2][Alpha] in [Ca.sup.2+] channel regulation using [G.sub.i2][Alpha] gene knockout mouse ventricular myocytes. The whole cell voltage-clamp technique was used to study the effects of the muscarinic agonist carbachol (CCh) and the [Beta]-adrenergic agonist isoproterenol (Iso) on cardiac L-type [Ca.sup.2+] currents in both 129Sv wild-type (WT) and [G.sub.i2][Alpha] gene knockout ([G.sub.i2][Alpha]-/-) mice. Perfusion with CCh significantly inhibited the [Ca.sup.2+] current in WT cells, and this effect was reversed by adding atropine to the CCh-containing solution. In contrast, CCh did not affect [Ca.sup.2+] currents in [G.sub.i2][Alpha]-/- ventricular myocytes. Addition of CCh to Iso-containing solutions attenuated the Iso-stimulated [Ca.sup.2+] current in WT cardiomyocytes but not in [G.sub.i2][Alpha]-/- cells. These findings demonstrate that, whereas the Iso-[G.sub.s][Alpha] signal pathway is intact in [G.sub.i2][Alpha] gene knockout mouse hearts, these cells lack the inhibitory regulation of [Ca.sup.2+] channels by CCh. Therefore, [G.sub.i2][Alpha] is necessary for the muscarinic regulation of [Ca.sup.2+] channels in the mouse heart. Further studies are needed to delineate the possible interaction of [G.sub.i] and other cell signaling proteins and to clarify the level of interaction of G protein-coupled regulation of L-type [Ca.sup.2+] current in the heart. [Ca.sup.2+] current regulation; gene inactivation; signal transduction

Homologues of Drosophilia transient receptor potential (TRP) have been proposed to be unitary subunits of plasma membrane ion channels that are activated as a consequence of active or passive depletion of [Ca.sup.2+] stores. In agreement with this hypothesis, cells expressing TRPs display novel [Ca.sup.2+]-permeable cation channels that can be activated by the inositol 1,4,5-trisphosphate receptor (IP3R) protein. Expression of TRPs alters cells in many ways, including up-regulation of IP3Rs not coded for by TRP genes, and proof that TRP forms channels of these and other cells is still missing. Here, we document physical interaction of TRP and IP3R by coimmunoprecipitation and glutathione S-transferase-pulldown experiments and identify two regions of IP3R, F2q and F2g, that interact with one region of TRP, C7. These interacting regions were expressed in cells with an unmodified complement of TRPs and IP3Rs to study their effect on agonist- as well as store depletion-induced [Ca.sup.2+] entry and to test for a role of their respective binding partners in [Ca.sup.2+] entry. C7 and an F2q-containing fragment of IP3R decreased both forms of [Ca.sup.2+] entry. In contrast, F2g enhanced the two forms of [Ca.sup.2+] entry. We conclude that store depletion-activated [Ca.sup.2+] entry occurs through channels that have TRPs as one of their normal structural components, and that these channels are directly activated by IP3Rs. IP3Rs, therefore, have the dual role of releasing [Ca.sup.2+] from stores and activating [Ca.sup.2+] influx in response to either increasing IP3 or decreasing luminal [Ca.sup.2+].