G protein modulation of recombinant P/Q-type calcium channels by regulators of G protein signalling proteins.

1. Fast synaptic transmission is triggered by the activation of presynaptic Ca2+ channels which can be inhibited by Gbetagamma subunits via G protein-coupled receptors (GPCR). Regulators of G protein signalling (RGS) proteins are GTPase-accelerating proteins (GAPs), which are responsible for >100-fold increases in the GTPase activity of G proteins and might be involved in the regulation of presynaptic Ca2+ channels. In this study we investigated the effects of RGS2 on G protein modulation of recombinant P/Q-type channels expressed in a human embryonic kidney (HEK293) cell line using whole-cell recordings. 2. RGS2 markedly accelerates transmitter-mediated inhibition and recovery from inhibition of Ba2+ currents (IBa) through P/Q-type channels heterologously expressed with the muscarinic acetylcholine receptor M2 (mAChR M2). 3. Both RGS2 and RGS4 modulate the prepulse facilitation properties of P/Q-type Ca2+ channels. G protein reinhibition is accelerated, while release from inhibition is slowed. These kinetics depend on the availability of G protein alpha and betagamma subunits which is altered by RGS proteins. 4. RGS proteins unmask the Ca2+ channel beta subunit modulation of Ca2+ channel G protein inhibition. In the presence of RGS2, P/Q-type channels containing the beta2a and beta3 subunits reveal significantly altered kinetics of G protein modulation and increased facilitation compared to Ca2+ channels coexpressed with the beta1b or beta4 subunit.

G-protein mediated gating of inward-rectifier K+ channels.

G-protein regulated inward-rectifier potassium channels (GIRK) are part of a superfamily of inward-rectifier K+ channels which includes seven family members. To date four GIRK subunits, designated GIRK1-4 (also designated Kir3.1-4), have been identified in mammals, and GIRK5 has been found in Xenopus oocytes. GIRK channels exist in vivo both as homotetramers and heterotetramers. In contrast to the other mammalian GIRK family members, GIRK1 can not form functional channels by itself and has to assemble with GIRK2, 3 or 4. As the name implies, GIRK channels are modulated by G-proteins; they are also modulated by phosphatidylinositol 4,5-bisphosphate, intracellular sodium, ethanol and mechanical stretch. Recently a family of GTPase activating proteins known as regulators of G-protein signaling were shown to be the missing link for the fast deactivation kinetics of GIRK channels in native cells, which contrast with the slow kinetics observed in heterologously expressed channels. GIRK1, 2 and 3 are highly abundant in brain, while GIRK4 has limited distribution. Here, GIRK1/2 seems to be the predominant heterotetramer. In general, neuronal GIRK channels are involved in the regulation of the excitability of neurons and may contribute to the resting potential. Interestingly, only the GIRK1 and 4 subunits are distributed in the atrial and sinoatrial node cells of the heart and are involved in the regulation of cardiac rate. Our main objective of this review is to assess the current understanding of the G-protein modulation of GIRK channels and their physiological importance in mammals.

Regulation of GIRK channel deactivation by Galpha(q) and Galpha(i/o) pathways.

G protein regulated inward rectifying potassium channels (GIRKs) are activated by G protein coupled receptors (GPCRs) via the G protein betagamma subunits. However, little is known about the effects of different GPCRs on the deactivation kinetics of transmitter-mediated GIRK currents. In the present study we investigated the influence of different GPCRs in the presence and absence of RGS proteins on the deactivation kinetics of GIRK channels by coexpressing the recombinant protein subunits in Xenopus oocytes. The stimulation of both G(i/o)- and G(q)-coupled pathways accelerated GIRK deactivation. GIRK currents deactivated faster upon stimulation of G(i/o)- and G(q)-coupled pathways by P(2)Y(2) receptors (P(2)Y(2)Rs) than upon activation of the G(i/o)-coupled pathway alone via muscarinic acetylcholine receptor M2 (M(2) mAChRs). This acceleration was found to be dependent on phospholipase C (PLC) and protein kinase C (PKC) activities and intracellular calcium. With the assumption that RGS2 has a higher affinity for Galpha(q) than Galpha(i/o), we demonstrated that the deactivation kinetics of GIRK channels can be differentially regulated by the relative amount of RGS proteins. These data indicate that transmitter-mediated deactivation of GIRK currents is modulated by crosstalk between G(i/o)- and G(q)-coupled pathways.

Synaptic localization and presynaptic function of calcium channel beta 4-subunits in cultured hippocampal neurons.

Neurotransmitter release is triggered by the influx of Ca(2+) into the presynaptic terminal through voltage gated Ca(2+)-channels. The shape of the presynaptic Ca(2+) signal largely determines the amount of released quanta and thus the size of the synaptic response. Ca(2+)-channel function is modulated in particular by the auxiliary beta-subunits that interact intracellularly with the pore-forming alpha(1)-subunit. Using retrovirus-mediated gene transfer in cultured hippocampal neurons, we demonstrate that functional GFP-beta(4) constructs colocalize with the synaptic vesicle marker synaptobrevin II and endogenous P/Q-type channels, indicating that beta(4)-subunits are localized to synaptic sites. Costaining with the dendritic marker MAP2 revealed that the beta(4)-subunit is transported to dendrites as well as axons. The nonconserved amino- and carboxyl-termini of the beta(4)-subunit were found to target the protein to the synapse. Physiological measurements in autaptic hippocampal neurons infected with green fluorescent protein (GFP)-beta(4) revealed an increase in both excitatory post-synaptic current amplitude and paired pulse facilitation ratio, whereas the GFP-beta(4) mutant, GFP-beta(4)(Delta50-407), which demonstrated a cytosolic localization pattern, did not alter these synaptic properties. In summary, our data suggest a pre-synaptic function of the Ca(2+)-channel beta(4)-subunit in synaptic transmission.

New roles for RGS2, 5 and 8 on the ratio-dependent modulation of recombinant GIRK channels expressed in Xenopus oocytes.

1. The activation of G protein-regulated inward rectifying potassium (GIRK) channels is modulated by G protein-coupled receptors (GPCRs) via the G protein betagamma subunits and is accelerated by regulators of G protein signalling (RGS). In the present study we investigated the ratio dependence of receptor-mediated activation and deactivation and the influence of new members of the RGS protein family on GIRK currents by coexpressing the recombinant protein subunits in Xenopus oocytes and further analysis of the whole cell currents. 2. The activation of GIRK channels by the muscarinic acetylcholine receptor M2 (M2 mAChR) is strongly dependent on the ratio of receptor to channel in Xenopus oocytes. The increase and on-rate of the amplified current is affected by this ratio. An excess of receptor over channel is necessary for current amplification, while the reverse excess of channel over receptor abolishes the effect. 3. The speed of receptor-mediated activation of GIRK currents is accelerated for a high ratio of receptor to channel, while the time of deactivation is independent of this ratio. 4. Coexpression of RGS2, 5 and 8 accelerates the speed for ACh-mediated activation and deactivation of GIRK1/2 and GIRK1/4 currents. Thereby the receptor/channel/RGS ratio determines the amount of current amplification. 5. Bordetella pertussis toxin completely abolished ACh-mediated current amplification of GIRK channels coexpressed with or without RGS2. 6. Two single point mutations in the RGS2 protein (RGS2(N109S) and RGS2(L180F)) reduced the acceleration of current amplification after ACh application on GIRK1/4 channels compared with RGS2 wild-type protein.

Patterning neuronal connections by chemorepulsion: the semaphorins.

Axonal growth cones navigate long distances along specific pathways to establish complex patterns of neuronal connections. A growing number of signals have been identified that participate in these steering decisions. This review will concentrate on a large and growing family of chemorepellents, the semaphorins. This family contains both secreted and membrane-bound proteins expressed in many neuronal and non-neuronal tissues of invertebrates and vertebrates. Ongoing studies have given us a better understanding of how their highly conserved signalling system is involved in patterning neuronal connections.

Coupling of epidermal growth factor (EGF) with the antiproliferative activity of cAMP induces neuronal differentiation.

Nerve growth factor (NGF) functions as a progression factor with both mitogenic and antimitogenic activities. When PC12 cells are treated with NGF, they advance to the G1 stage of the cell cycle before they differentiate. The correlation between cessation of proliferation and differentiation suggests that the antimitotic activity of NGF may be obligatory for differentiation. Although epidermal growth factor- (EGF) and NGF-treated PC12 cells share several common properties, including activation of the mitogen-activated protein (MAP) kinase pathway and induction of immediate early genes, EGF is mitogenic for PC12 cells and does not normally stimulate differentiation. However, combinations of EGF and low levels of cAMP stimulate differentiation even though neither agent alone does (Mark, M. D., Liu, Y., Wong, S. T., Hinds, T. R., and Storm, D.R. (1995) J. Cell Biol. 130, 701-710). Since EGF is mitogenic for PC12 cells and differentiation may not occur until proliferation is inhibited, differentiation caused by cAMP and EGF may be due to the antiproliferative activity of cAMP. To test this hypothesis, we examined the effect of EGF or combinations of EGF and cAMP on PC12 cell proliferation. EGF alone stimulated proliferation of PC12 cells and increased the levels of several cell cycle progression factors including cdk2, cdk4, and cyclin B1. Cyclic AMP inhibited the EGF-stimulated increases in cell cycle progression factors as well as proliferation. Other antiproliferative agents including rapamycin, mimosine, and nitric oxide agonists also synergized with EGF to stimulate differentiation. These data indicate that the coupling of antiproliferative signals with EGF modifies the biological properties of EGF and converts it to a differentiating growth factor.

Antiproliferative activity of KCl contributes to EGF-induced neurite outgrowth in PC12 cells.

Combinations of epidermal growth factor (EGF) and KCl stimulate differentiation in PC12 cells, independent of extracellular calcium [Mark et al., Stimulation of neurite outgrowth in PC12 cells by EGF and KCl depolarization: a Ca2+-independent phenomenon, J. Cell Biol., 130 (1995) 701-710]. Since EGF is a proliferative agent that normally does not stimulate differentiation of PC12 cells, we hypothesize that KCl plus EGF may cause differentiation because of the anti-proliferative activity of KCl. Here we report that treatment of PC12 cells with KCl plus EGF resulted in a significant decrease in proliferation and DNA synthesis compared with cells treated with EGF alone. In addition, KCl significantly reduced the EGF-induced expression of cell cycle progression factors cdk2, cdk4, cyclin B1 and PCNA. These data suggest that the anti-proliferative activity of KCl may convert EGF from a proliferative factor to a progression factor.

Stimulation of neurite outgrowth in PC12 cells by EGF and KCl depolarization: a Ca(2+)-independent phenomenon.

MAP kinase activity is necessary for growth factor induction of neurite outgrowth in PC12 cells. Although NGF and EGF both stimulate MAP kinase activity, EGF does not stimulate neurite extension. We report that EGF, in combination with KCl, stimulates neurite outgrowth in PC12 cells. This phenomenon was independent of intracellular Ca2+ increases and not due to enhancement of MAP kinase activity over that seen with EGF alone. However, EGF plus KCl increased intracellular cAMP, and other cAMP elevating agents acted synergistically with EGF to promote neurite outgrowth. Stimulation of neurite outgrowth by cAMP and EGF was blocked by inhibitors of transcription suggesting that synergistic regulation of transcription by the cAMP and MAP kinase pathways may stimulate neurite growth.

Multiple strictures in Crohn's disease of the small bowel: a benign variant.

Involvement of both jejunum and ileum is uncommon in Crohn's disease of the small bowel. We report five patients with multiple strictures of the small bowel associated with one or more intervening segments of dilated bowel. A diagnosis of Crohn's disease was delayed because none of the patients experienced diarrhea. Despite the early radiologic appearance of extensive small bowel disease, only three patients have required surgery, a limited surgical resection of 65-75 cm was possible, and long-term prognosis has been favorable.