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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 (1,2). Similar conditions of nutrient limitation [...]

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.

The G protein [G.sub.o] is highly expressed in neurons and mediates effects of a group of rhodopsin-like receptors that includes the opioid, [[Alpha].sub.2]-adrenergic, M2 muscarinic, and somatostatin receptors. In vitro, [G.sub.o] is also activated by growth cone-associated protein of [M.sub.r] 43,000 (GAP43) and the Alzheimer amyloid precursor protein, but it is not known whether this occurs in intact cells. To learn about the roles that [G.sub.o] may play in intact cells and whole body homeostasis, we disrupted the gene encoding the a subunits of [G.sub.o] in embryonic stem cells and derived [G.sub.o]-deficient mice. Mice with a disrupted [[Alpha].sub.o] gene ([[Alpha].sub.o]-/- mice) lived but had an average half-life of only about 7 weeks. No [G.sub.o][Alpha] was detectable in homogenates of [[Alpha].sub.o]-/- mice by ADP-ribosylation with pertussis toxin. At the cellular level, inhibition of cardiac adenylyl cyclase by carbachol (50-55% at saturation) was unaffected, but inhibition of [Ca.sup.2+] channel currents by opioid receptor agonist in dorsal root ganglion cells was decreased by 30%, and in 25% of the [[Alpha].sub.o]-/- cells examined, the [Ca.sup.2+] channel was activated at voltages that were 13.3 [+ or -] 1.7 mV lower than in their counterparts. Loss of [[Alpha].sub.o] was not accompanied by appearance of significant amounts of active free [Beta][Gamma] dimers (prepulse test). At the level of the living animal, [G.sub.o]-deficient mice are hyperalgesic (hot-plate test) and display a severe motor control impairment (falling from rotarods and 1-inch wide beams). In spite of this deficiency, [[Alpha].sub.o]-/- mice are hyperactive and exhibit a turning behavior that has them running in circles for hours on end, both in cages and in open-field tests. Except for one, all [[Alpha].sub.o]-/- mice turned only counterclockwise. These findings indicate that [G.sub.o] plays a major role in motor control, in motor behavior, and in pain perception and also predict involvement of [G.sub.o] in [Ca.sup.2+] channel regulation by all unknown mechanism.

During the last 2 years, our laboratory has worked on the elucidation of the molecular basis of capacitative calcium entry (CCE) into cells. Specifically, we tested the hypothesis that CCE channels are formed of subunits encoded in genes related to the Drosophila trp gene. The first step in this pursuit was to search for mammalian trp genes. We found not one but six mammalian genes and cloned several of their cDNAs, some in their full length. As assayed in mammalian cells, overexpression of some mammalian Trps increases CCE, while expression of partial trp cDNAs in antisense orientation can interfere with endogenous CCE. These findings provided a firm connection between CCE and mammalian Trps. This article reviews the known forms of CCE and highlights unanswered questions in our understanding of intracellular [Ca.sup.2+] homeostasis and the physiological roles of CCE.

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.

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

We reported previously that Go-deficient mice develop severe neurological defects that include hyperalgesia, a generalized tremor, lack of coordination, and a turning syndrome somewhat reminiscent of unilateral lesions of the dopaminergic nigro-striatal pathway. By using frozen coronal sections of serially sectioned brains of normal and Go-deficient mice, we studied the ability of several G protein coupled receptors to promote binding of GTP[Gamma]S to G proteins and the ability of GTP to promote a shift in the affinity of D2 dopamine receptor for its physiologic agonist dopamine. We found a generalized, but not abolished reduction in agonist-stimulated binding of GTP[Gamma]S to frozen brain sections, with no significant left-right differences. Unexpectedly, the ability of GTP to regulate the binding affinity of dopamine to D2 receptors (as seen in in situ [[sup.35]S]sulpiride displacement curves) that was robust in control mice, was absent in Go-deficient mice. The data suggest that most of the effects of the Gi/Go-coupled D2 receptors in the central nervous system are mediated by Go instead of Gi1, Gi2, or Gi3. In agreement with this, the effect of GTP on dopamine binding to D2 receptors in double Gi1 plus Gi2- and Gi1 plus Gi3-deficient mice was essentially unaffected. G protein | knockout mice | homologous recombination | GTP shift | sulpiride

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+].