The neuropeptide FMRFa both inhibits and enhances the Ca2+ current in dissociated Helix neurons via independent mechanisms

1991 ◽  
Vol 65 (6) ◽  
pp. 1517-1527 ◽  
Author(s):  
J. L. Yakel
Keyword(s):  
G Protein ◽  
Cell Voltage ◽  
Inhibitory Response ◽  
Central Neurons ◽  
S 100 ◽  
Helix Neurons ◽  
20 Nm ◽  
Gamma S ◽  
Ca2 Channels ◽  
Rate Of Activation

1. The modulation of the voltage-activated Ca2+ current by the neuropeptide Phe-Met-Arg-Phe-NH2 (FMRFa) was investigated in dissociated central neurons from Helix aspersa using whole-cell voltage-clamp recording techniques. External Ba2+ was always used as the charge carrier in this study, and the intracellular Ca2+ concentration was buffered to 20 nM with ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA). 2. Run-down of the Ca2+ currents was not a problem as long as the neurons were dialyzed with a patch electrode filling solution containing ATP (1 or 2 mM). In ATP-dialyzed neurons, the rate of inactivation of the calcium current increased with time without any significant change in the rate of activation. However, when neurons were dialyzed with guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S; 100 microM; with ATP), the rate of inactivation decreased with time. There was no effect of GTP gamma S on the rate of activation of the Ca2+ current. This suggests that guanosine 5'-triphosphate (GTP)-binding proteins (G proteins) are able to modulate the rate of inactivation of the Ca2+ current in Helix neurons. 3. FMRFa both decreased and enhanced the amplitude of the Ca2+ current in these neurons. This inhibition was observed in most neurons, while the enhancement was observed in 20% of the neurons. Although the enhancement usually was preceded by the inhibitory response, sometimes the enhancement was observed separately. 4. The FMRFa-induced inhibition of the Ca2+ current usually consisted of a decrease in both the amplitude and the rate of inactivation of this current, effects that were reduced as the membrane potential was stepped to more depolarized potentials. A pertussis toxin (PTX)-sensitive G protein mediated this response, whereas no evidence was found to suggest the involvement of any known intracellular messenger. Therefore this inhibition may have resulted from a direct coupling between the FMRFa receptor and the Ca2+ channels via a PTX-sensitive G protein. 5. Arachidonic acid (100 microM) irreversibly reduced the amplitude of the Ca2+ current, but it did not alter the relative inhibition of this current by FMRFa. 6. The FMRFa-induced enhancement of the Ca2+ current was difficult to study because it was observed infrequently, and was rarely observed independently of the FMRFa-induced inhibitory response. In addition, the ability of FMRFa to enhance this current usually disappeared with time.(ABSTRACT TRUNCATED AT 400 WORDS)

Pancreatic polypeptide inhibits calcium channels in rat sympathetic neurons via two signaling pathways

1995 ◽  
Vol 73 (3) ◽  
pp. 1323-1328 ◽  
Author(s):  
L. P. Wollmuth ◽  
M. S. Shapiro ◽  
B. Hille
Keyword(s):  
G Protein ◽  
Pertussis Toxin ◽  
Pancreatic Polypeptide ◽  
Sympathetic Neurons ◽  
Whole Cell ◽  
Adult Rat ◽  
Voltage Dependent ◽  
Cervical Ganglion ◽  
Gamma S ◽  
Ca2 Channels

1. We studied modulation of N-type Ca2+ channels in adult rat superior cervical ganglion (SCG) neurons by pancreatic polypeptide (PP) using whole cell clamp. In large (> 20 pF) SCG neurons, PP inhibited ICa (35 +/- 2%, mean +/- SE) in a concentration-dependent fashion, with one-half maximal inhibition at 19 nM. 2. One-third of the inhibition was blocked by pertussis toxin, about one-half was blocked by N-ethylmaleimide (NEM) treatments, and about one-half was voltage dependent. The NEM-insensitive component of the PP inhibition was voltage independent and not significantly blocked by intracellular Ca2+ chelators. 3. The NEM-insensitive component was only weakly attenuated by GDP-beta-S, and moderately reversible with guanosine 5'-triphosphate (GTP)-gamma-S, in the whole cell pipette, leaving open the possibility that it is not mediated by a G protein. 4. Hence, PP inhibits ICa via two mechanisms: one G-protein-mediated and the other possibly G-protein independent. The former pathway is sensitive to pertussis toxin (PTX) and NEM, voltage dependent, and shared by several other transmitters in these cells. The latter pathway is PTX-and NEM-insensitive, not voltage dependent, and not affected by the presence of intracellular Ca2+ chelators.

Ca2+ channel blockers distinguish between G protein-coupled pharmacomechanical Ca2+ release and Ca2+ sensitization

1991 ◽  
Vol 260 (2) ◽  
pp. C364-C370 ◽  
Author(s):  
S. Kobayashi ◽  
M. C. Gong ◽  
A. V. Somlyo ◽  
A. P. Somlyo
Keyword(s):  
Phospholipase C ◽  
G Protein ◽  
Smooth Muscles ◽  
Channel Blockers ◽  
Alpha Toxin ◽  
Adrenergic Agonist ◽  
Pharmacomechanical Coupling ◽  
Gamma S ◽  
G Protein Coupled ◽  
Ca2 Channels

The effects of Ca2+ channel blockers on two modes of G protein-mediated pharmacomechanical coupling, Ca2+ release and modulation of Ca2+ sensitivity of the contractile apparatus, were investigated. Smooth muscles were permeabilized with Staphylococcal alpha-toxin or with beta-escin to avoid effects due to block of sarcolemmal Ca2+ channels, while retaining receptor/G protein coupling. In permeabilized portal vein smooth muscle, verapamil and nifedipine inhibited Ca2+ release induced by an alpha 1-adrenergic agonist (phenylephrine) and by guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S), but not that induced by inositol 1,4,5-trisphosphate (InsP3). These Ca2+ channel blockers also did not block the phenylephrine- or GTP gamma S-induced force development at constant cytoplasmic Ca2+ ("Ca2+ sensitization"). An alpha 1-blocker (prazosin) inhibited both the Ca2(+)-releasing and Ca2(+)-sensitizing effects of phenylephrine, but not those of GTP gamma S, nor did it block InsP3-induced Ca2+ release. We conclude that Ca2+ channel blockers selectively uncouple the Ca2(+)-releasing, but not the Ca2(+)-sensitizing, component of pharmacomechanical coupling. These findings raise the possibility that pharmacomechanical Ca2+ release may be modulated by dihydropyridine binding proteins at the level of G proteins/phospholipase C and also indicate a divergence of the Ca2(+)-releasing and Ca2(+)-sensitizing effects at some step prior to phospholipase C.

scholarly journals Reconstitution of the ATP-sensitive potassium channel of skeletal muscle. Activation by a G protein-dependent process.

1989 ◽  
Vol 94 (3) ◽  
pp. 445-463 ◽  
Author(s):  
L Parent ◽  
R Coronado
Keyword(s):  
Skeletal Muscle ◽  
G Protein ◽  
Muscle Activation ◽  
Open Channel ◽  
Single Channel ◽  
Rabbit Skeletal Muscle ◽  
S 100 ◽  
Internal Side ◽  
Gamma S ◽  
Channel Properties

Potassium channels inhibited by adenosine-5'-trisphosphate, K(ATP), found in the transverse tubular membrane of rabbit skeletal muscle were studied using the planar bilayer recording technique. In addition to the single-channel properties of K(ATP) we report its regulation of Mg2+ and by the guanosine-5'-trisphosphate analogue, GTP-y(gamma)-S. The K(ATP) channel (a) has a conductance of 67 pS in 250 mM internal, 50 mM external KCl, and rectifies weakly at holding potentials more positive than 50 mV, (b) is not activated by internal Ca2+ or membrane depolarization, (c) has a permeability ratio PK/PNa greater than 50, and (d) is inhibited by millimolar internal ATP. Activity of K(ATP), measured as open channel probability as a function of time, was unstable at all holding potentials and decreases continuously within a few minutes after a recording is initiated. After a decrease in activity, GTP-y-S (100 microM) added to the internal side reactivated K(ATP) channels but only transiently. In the presence of internal 1 mM Mg2+, GTP-y-S produced a sustained reactivation lasting 20-45 min. Incubation of purified t-tubule vesicles with AlF4 increased the activity of K(ATP) channels, mimicking the effect of GTP-y-S. The effect of AlF4 and the requirement of GTP-y-S plus Mg2+ for sustained channel activation suggests that a nucleotide-binding G protein regulates ATP-sensitive K channels in the t-tuble membrane of rabbit skeletal muscle.

scholarly journals Ion permeation through 5-hydroxytryptamine-gated channels in neuroblastoma N18 cells.

1990 ◽  
Vol 96 (6) ◽  
pp. 1177-1198 ◽  
Author(s):  
J Yang
Keyword(s):  
Single Channel ◽  
Cell Voltage ◽  
Ionic Currents ◽  
Organic Cations ◽  
Fluctuation Analysis ◽  
Divalent Ions ◽  
Apparent Permeability ◽  
S 100 ◽  
Specific Antagonist ◽  
Gamma S

Ionic currents induced by 5-hydroxytryptamine (5-HT) in cultured neuroblastoma N18 cells were studied using whole-cell voltage clamp. The response was blocked by 1-10 nM 5-HT3 receptor-specific antagonists MDL 7222 or ICS 205-930, but not by 1 microM 5-HT1/5-HT2 receptor antagonist spiperone or 5-HT2 receptor-specific antagonist ketanserin. These 5-HT3 receptors seem to be ligand-gated channels because the response (a) did not require internal ATP or GTP, (b) persisted with long internal dialysis of CsF (90 mM), A1F4- (100 microM), or GTP gamma S (100 microM), and (c) with ionophoretic delivery of 5-HT developed with a delay of less than 10 ms and rose to a peak in 34-130 ms. Fluctuation analysis yielded an apparent single-channel conductance of 593 fS. The relative permeabilities of the channel for a variety of ions were determined from reversal potentials. The channel was only weakly selective among small cations, with permeability ratios PX/PNa of 1.22, 1.10, 1.01, 1.00, and 0.99 for Cs+, K+, Li+, Na+, and Rb+, and 1.12, 0.79, and 0.73 for Ca2+, Ba2+, and Mg2+ (when studied in mixtures of 20 mM divalent ions and 120 mM N-methyl-D-glucamine). Apparent permeability ratios for the divalent ions decreased as the concentration of divalent ions was increased. Small monovalent organic cations were highly permeant. Large organic cations such as Tris and glucosamine were measurably permeant with permeability ratios of 0.20 and 0.08, and N-methyl-D-glucamine was almost impermeant. Small anions, NO3-, Cl-, and F-, were slightly permeant with permeability ratios of 0.08, 0.04, and 0.03. The results indicate that the open 5-HT3 receptor channel has an effective minimum circular pore size of 7.6 A and that ionic interactions in the channel may involve negative charges near the pore mouth.

scholarly journals Epidermal growth factor-induced phosphoinositide hydrolysis in permeabilized 3T3 cells: lack of guanosine triphosphate dependence and inhibition by tyrosine-containing peptides.

1990 ◽  
Vol 1 (9) ◽  
pp. 615-620 ◽  
Author(s):  
G F Verheijden ◽  
I Verlaan ◽  
J Schlessinger ◽  
W H Moolenaar
Keyword(s):  
Growth Factor ◽  
Epidermal Growth Factor ◽  
G Protein ◽  
Egf Receptor ◽  
Phosphoinositide Hydrolysis ◽  
3T3 Cells ◽  
Guanosine Triphosphate ◽  
Intact Cells ◽  
Epidermal Growth ◽  
Gamma S

The possible involvement of a stimulatory guanosine triphosphate (GTP)-binding (G) protein in epidermal growth factor (EGF)-induced phosphoinositide hydrolysis has been investigated in permeabilized NIH-3T3 cells expressing the human EGF receptor. The mitogenic phospholipid lysophosphatidate (LPA), a potent inducer of phosphoinositide hydrolysis, was used as a control stimulus. In intact cells, pertussis toxin partially inhibits the LPA-induced formation of inositol phosphates, but has no effect on the response to EGF. In cells permeabilized with streptolysin-O, guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) dramatically increases the initial rate of inositol phosphate formation induced by LPA. In contrast, activation of phospholipase C (PLC) by EGF occurs in a GTP-independent manner. Guanine 5'-O-(2-thiodiphosphate) (GDP beta S) which keeps G proteins in their inactive state, blocks the stimulation by LPA and GTP gamma S, but fails to affect the EGF-induced response. Tyrosine-containing substrate peptides, when added to permeabilized cells, inhibit EGF-induced phosphoinositide hydrolysis without interfering with the response to LPA and GTP gamma S. These data suggest that the EGF receptor does not utilize an intermediary G protein to activate PLC and that receptor-mediated activation of effector systems can be inhibited by exogenous substrate peptides.

scholarly journals Intracellular Na+inhibits voltage-dependent N-type Ca2+channels by a G protein βγ subunit-dependent mechanism

2004 ◽  
Vol 556 (1) ◽  
pp. 121-134 ◽  
Author(s):  
Yakov Blumenstein ◽  
Olexandr P. Maximyuk ◽  
Natalia Lozovaya ◽  
Natalia M. Yatsenko ◽  
Nataly Kanevsky ◽  
...  
Keyword(s):  
G Protein ◽  
Voltage Dependent ◽  
Ca2 Channels ◽  
Dependent Mechanism

scholarly journals Differential Interactions of the C terminus and the Cytoplasmic I-II Loop of Neuronal Ca2+Channels with G-protein α and βγ Subunits

1998 ◽  
Vol 273 (28) ◽  
pp. 17595-17603 ◽  
Author(s):  
Taiji Furukawa ◽  
Reiko Miura ◽  
Yasuo Mori ◽  
Mark Strobeck ◽  
Kazuyuki Suzuki ◽  
...  
Keyword(s):  
G Protein ◽  
C Terminus ◽  
Ca2 Channels

scholarly journals Mechanism of acetylcholine-induced inhibition of Ca current in bullfrog atrial myocytes.

1990 ◽  
Vol 96 (4) ◽  
pp. 865-885 ◽  
Author(s):  
T Nakajima ◽  
S Wu ◽  
H Irisawa ◽  
W Giles
Keyword(s):  
Adenylate Cyclase ◽  
Binding Protein ◽  
Cell Voltage ◽  
Inhibitory Effect ◽  
Atrial Myocytes ◽  
Ca Current ◽  
Gtp Binding Protein ◽  
Gtp Binding ◽  
Subsequent Application ◽  
Gamma S

The mechanism of the anti-beta-adrenergic action of acetylcholine (ACh) on Ca current, ICa, was examined using the tight-seal, whole-cell voltage clamp technique in single atrial myocytes from the bullfrog. Both isoproterenol (ISO) and forskolin increased ICa dose dependently. After ICa had been enhanced maximally by ISO (10(-6) M), subsequent application of forskolin (50 microM) did not further increase ICa, suggesting that ISO and forskolin increase ICa via a common biochemical pathway, possibly by stimulation of adenylate cyclase. ACh (10(-5) M) completely inhibited the effect of low doses of forskolin (2 x 10(-6) M), as well as ISO, but it failed to block the effects of high doses of forskolin (greater than 5 x 10(-5) M). Intracellular application of cyclic AMP (cAMP) also increased ICa. ACh (10(-5) M) failed to inhibit this cAMP effect, indicating that the inhibitory action of ACh occurs at a site proximal to the production of cAMP. ACh (10(-5) M) also activated an inwardly rectifying K+ current IK(ACh). Intracellular application of a nonhydrolyzable GTP analogue, GTP gamma S (5 X 10(-4) M), activated IK(ACh) within several minutes; subsequent application of ACh (10(-5) M) did not increase IK(ACh) further. These results demonstrate that a GTP-binding protein coupled to these K+ channels can be activated maximally by GTP gamma S even in the absence of ACh. Intracellular application of GTP gamma S also strongly inhibited the effect of ISO on ICa in the absence of ACh. Pertussis toxin (IAP) completely prevented both the inhibitory effect of ACh on ICa and the ACh-induced activation of IK(ACh). GTP gamma S (50 microM-1 mM) alone did not increase ICa significantly; however, when ISO was applied first, GTP gamma S (5 x 10(-4) M) gradually inhibited the ISO effect on ICa. These results indicate that ACh antagonizes the effect of ISO on ICa via a GTP-binding protein (Gi and/or Go). This effect may be mediated through a direct inhibition by the alpha-subunit of Gi which is coupled to the adenylate cyclase.

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