The Small GTP-Binding Protein RhoA Regulates a Delayed Rectifier Potassium Channel

AbstractTyrosine kinases activated by G protein–coupled receptors can phosphorylate and thereby suppress the activity of the delayed rectifier potassium channel Kv1.2. Using a yeast two-hybrid screen, we identified the small GTP-binding protein RhoA as a necessary component in this process. Coimmunoprecipitation experiments confirmed that RhoA associates with Kv1.2. Electrophysiological analyses revealed that overexpression of RhoA markedly reduced the basal current generated by Kv1.2 expressed in Xenopus oocytes. Furthermore, in 293 cells expressing Kv1.2 and m1 muscarinic acetylcholine receptors, inactivating RhoA using C3 exoenzyme blocked the ability of m1 receptors to suppress Kv1.2 current. Therefore, these results demonstrate that RhoA regulates Kv1.2 activity and is a central component in the mechanism of receptor-mediated tyrosine kinase–dependent suppression of Kv1.2

Cholinergic suppression: A postsynaptic mechanism of long-term associative learning

Food avoidance learning in the mollusc Pleurobranchaea entails reduction in the responsiveness of key brain interneurons in the feeding neural circuitry, the paracerebral feeding command interneurons (PCNs), to the neurotransmitter acetylcholine (AcCho). Food stimuli applied to the oral veil of an untrained animal depolarize the PCNs and induce the feeding motor program (FMP). Atropine (a muscarinic cholinergic antagonist) reversibly blocks the food-induced depolarization of the PCNs, implicating AcCho as the neurotransmitter mediating food detection. AcCho applied directly to PCN somata depolarizes them, indicating that the PCN soma membrane contains AcCho receptors and induces the FMP in the isolated central nervous system preparation. The AcCho response of the PCNs is mediated by muscariniclike receptors, since comparable depolarization is induced by muscarinic agonists (acetyl-ß -methylcholine, oxotremorine, pilocarpine), but not nicotine, and blocked by muscarinic antagonists (atropine, trifluoperazine). The nicotinic antagonist hexamethonium, however, blocked the AcCho response in four of six cases. When specimens are trained to suppress feeding behavior using a conventional food-avoidance learning paradigm (conditionally paired food and shock), AcCho applied to PCNs in the same concentration as in untrained animals causes little or no depolarization and does not initiate the FMP. Increasing the concentration of AcCho 10-100 times, however, induces weak PCN depolarization in trained specimens, indicating that learning diminishes but does not fully abolish AcCho responsiveness of the PCNs. This study proposes a cellular mechanism of long-term associative learning -- namely, postsynaptic modulation of neurotransmitter responsiveness in central neurons that could apply also to mammalian species

Kv1.3 channels in postganglionic sympathetic neurons: expression, function, and modulation

Kv1.3 channels are known to modulate many aspects of neuronal function. We tested the hypothesis that Kv1.3 modulates the function of postganglionic sympathetic neurons. RT-PCR, immunoblot, and immunohistochemical analyses indicated that Kv1.3 channels were expressed in these neurons. Immunohistochemical analyses indicated that Kv1.3 protein was localized to neuronal cell bodies, processes, and nerve fibers at sympathetic neurovascular junctions. Margatoxin (MgTX), a specific inhibitor of Kv1.3, was used to assess the function of the channel. Electrophysiological analyses indicated that MgTX significantly reduced outward currents [P < 0.05; n = 18 (control) and 15 (MgTX)], depolarized resting membrane potential, and decreased the latency to action potential firing [P < 0.05; n = 11 (control) and 13 (MgTX)]. The primary physiological input to postganglionic sympathetic neurons is ACh, which activates nicotinic and muscarinic ACh receptors. MgTX modulated nicotinic ACh receptor agonist-induced norepinephrine release (P < 0.05; n ≥ 6), and MgTX-sensitive current was suppressed upon activation of muscarinic ACh receptors with bethanechol (P < 0.05; n = 12). These data indicate that Kv1.3 affects the function of postganglionic sympathetic neurons, which suggests that Kv1.3 influences sympathetic control of cardiovascular function. Our data also indicate that modulation of Kv1.3 is likely to affect sympathetic control of cardiovascular function

FGT-1 Is a Mammalian GLUT2-Like Facilitative Glucose Transporter in <i>Caenorhabditis elegans</i> Whose Malfunction Induces Fat Accumulation in Intestinal Cells

<div><p><i>Caenorhabditis elegans</i> (<i>C. elegans</i>) is an attractive animal model for biological and biomedical research because it permits relatively easy genetic dissection of cellular pathways, including insulin/IGF-like signaling (IIS), that are conserved in mammalian cells. To explore <i>C. elegans</i> as a model system to study the regulation of the facilitative glucose transporter (GLUT), we have characterized the GLUT gene homologues in <i>C. elegans: fgt-1</i>, <i>R09B5.11, C35A11.4, F53H8.3, F48E3.2, F13B12.2, Y61A9LA.1, K08F9.1</i> and <i>Y37A1A.3</i>. The exogenous expression of these gene products in <i>Xenopus</i> oocytes showed transport activity to unmetabolized glucose analogue 2-deoxy-D-glucose only in FGT-1. The FGT-1-mediated transport activity was inhibited by the specific GLUT inhibitor phloretin and exhibited a Michaelis constant (<i>K</i>_<i>m</i>) of 2.8 mM. Mannose, galactose, and fructose were able to inhibit FGT-1<i>-</i>mediated 2-deoxy-D-glucose uptake (<i>P</i> < 0.01), indicating that FGT-1 is also able to transport these hexose sugars. A GFP fusion protein of FGT-1 was observed only on the basolateral membrane of digestive tract epithelia in <i>C. elegans</i>, but not in other tissues. FGT-1::eGFP expression was observed from early embryonic stages. The knockdown or mutation of <i>fgt-1</i> resulted in increased fat staining in both wild-type and <i>daf-2</i> (mammalian insulin receptor homologue) mutant animals. Other common phenotypes of IIS mutant animals, including dauer formation and brood size reduction, were not affected by <i>fgt-1</i> knockdown in wild-type or <i>daf</i>-2 mutants. Our results indicated that in <i>C. elegans</i>, FGT-1 is mainly a mammalian GLUT2-like intestinal glucose transporter and is involved in lipid metabolism.</p> </div

Amino acid sequence alignments of human GLUT1-4 and <i>C. elegans</i> FGT-1 and R09B5.11.

<p>A. Alignments of the deduced amino acid sequences of FGT-1, R09B5.11 and human GLUT1-4 were performed with the Clustal W program with open gap penalty = 10 and gap extension penalty = 0.05. Residues that are highlighted by a black shaded background represent absolutely conserved amino acids, and the gray shaded background indicates four or more conserved residues at those positions. Regions of presumed transmembrane domains (TM) [32] are indicated by numbered dashed lines, and the functionally important residues for glucose uptake activity are indicated by the letter “F” at the top of the sequence alignments. In addition, the highly conserved amino acids are shown on the bottom of the sequence alignment. B. Phylogenetic tree of the aligned sequences in (A) by the Clustal W program. Scale bar indicates relative branch lengths obtained from the Clustal W alignment result.</p

Structural schematic representation of GLUT candidate genes in <i>C. elegans</i>, compared with human GLUT4.

<p>The amino acid sequence of individual <i>C. elegans</i> genes was obtained from Wormbase (http://www.wormbase.org/). The blue boxes indicate the predicted transmembrane domains by Wormbase, and the dashed boxes in <i>R09B5.11</i> indicate the missing predicted transmembrane domains. Red filled circles indicate potential N-glycosylation sites that were predicted by NetNGlyc (http://www.cbs.dtu.dk/services/NetNGlyc/). Arrowheads indicate known functionally important residues that were found in human GLUT4: R92, R153, R333/4, and E393 [11]. The predicted conserved long loop 6 is indicated by red dashed circles.</p