scholarly journals Intracellular Cl− Dependence of Na-H Exchange in Barnacle Muscle Fibers under Normotonic and Hypertonic Conditions

1997 ◽  
Vol 110 (5) ◽  
pp. 629-639 ◽  
Author(s):  
Emilia M. Hogan ◽  
Bruce A. Davis ◽  
Walter F. Boron
Keyword(s):  
G Protein ◽  
Signal Transduction Pathway ◽  
Dose Dependence ◽  
Artificial Seawater ◽  
Hypertonic Solution ◽  
Barnacle Muscle ◽  
Barnacle Muscle Fibers ◽  
Exchange Flux ◽  
Exchange Activity ◽  
Barnacle Muscle Fiber

We previously showed that shrinking a barnacle muscle fiber (BMF) in a hypertonic solution (1,600 mosM/kg) stimulates an amiloride-sensitive Na-H exchanger. This activation is mediated by a G protein and requires intracellular Cl−. The purpose of the present study was to determine (a) whether Cl− plays a role in the activation of Na-H exchange under normotonic conditions (975 mosM/kg), (b) the dose dependence of [Cl−]i for activation of the exchanger under both normo- and hypertonic conditions, and (c) the relative order of the Cl−- and G-protein-dependent steps. We acid loaded BMFs by internally dialyzing them with a pH-6.5 dialysis fluid containing no Na+ and 0–194 mM Cl−. The artificial seawater bathing the BMF initially contained no Na+. After dialysis was halted, adding 50 mM Na+ to the artificial seawater caused an amiloride-sensitive pHi increase under both normo- and hypertonic conditions. The computed Na-H exchange flux (JNa-H) increased with increasing [Cl−]i under both normo- and hypertonic conditions, with similar apparent Km values (∼120 mM). However, the maximal JNa-H increased by nearly 90% under hypertonic conditions. Thus, activation of Na-H exchange at low pHi requires Cl− under both normo- and hypertonic conditions, but at any given [Cl−]i, JNa-H is greater under hyper- than normotonic conditions. We conclude that an increase in [Cl−]i is not the primary shrinkage signal, but may act as an auxiliary shrinkage signal. To determine whether the Cl−-dependent step is after the G-protein-dependent step, we predialyzed BMFs to a Cl−-free state, and then attempted to stimulate Na-H exchange by activating a G protein. We found that, even in the absence of Cl−, dialyzing with GTPγS or AlF3, or injecting cholera toxin, stimulates Na-H exchange. Because Na-H exchange activity was absent in control Cl−-depleted fibers, the Cl−-dependent step is at or before the G protein in the shrinkage signal-transduction pathway. The stimulation by AlF3 indicates that the G protein is a heterotrimeric G protein.

Shrinkage-induced activation of Na(+)-H+ exchange in barnacle muscle fibers

1994 ◽  
Vol 266 (6) ◽  
pp. C1744-C1753 ◽  
Author(s):  
B. A. Davis ◽  
E. M. Hogan ◽  
W. F. Boron
Keyword(s):  
Intracellular Ph ◽  
Muscle Fibers ◽  
Single Muscle ◽  
Extrusion Rate ◽  
Barnacle Muscle ◽  
Hypertonic Solutions ◽  
Acid Extrusion ◽  
Ph Microelectrodes ◽  
Barnacle Muscle Fibers ◽  
Exchange Activity

We examined the effect of shrinkage on Na(+)-H+ exchange in single muscle fibers at intracellular pH (pHi) values of 6.8, 7.2, and 7.6 using pH microelectrodes and internal dialysis. Under normotonic conditions (975 mosmol/kgH2O) at pHi 6.8, the amiloride-sensitive acid-extrusion rate (JAmil/s) averaged 17 microM/min. Exposure to hypertonic solutions (1,600 mosmol/kgH2O) increased JAmil/s to 304 microM/min at pHi 6.8. At pHi approximately 7.2 and 7.6, hypertonicity increased JAmil/s from approximately 0 to approximately 172 microM/min (pHi 7.2) and approximately 0 to approximately 90 microM/min (pHi 7.6). Thus, under normotonic conditions, Na(+)-H+ exchange activity is slight at pHi approximately 6.8 and virtually nil at higher pHi values. Shrinkage stimulated Na(+)-H+ exchange, more at low pHi values. We also examined the Cl- dependence of the Na(+)-H+ exchanger's response to shrinkage. Our results indicate that shrinkage-induced activation of Na(+)-H+ exchange requires Cl-, specifically intracellular Cl-. These results establish that shrinkage is both pHi dependent and requires intracellular Cl-.

Permeability of barnacle muscle fibers to water and nonelectrolytes

1976 ◽  
Vol 30 (1) ◽  
pp. 197-212
Author(s):  
Daniel F. Wolff ◽  
Osvaldo A. Alvarez ◽  
Fernando F. Vargas
Keyword(s):  
Muscle Fibers ◽  
Barnacle Muscle ◽  
Barnacle Muscle Fibers

scholarly journals G-Protein β Subunit of Cochliobolus heterostrophus Involved in Virulence, Asexual and Sexual Reproductive Ability, and Morphogenesis

2004 ◽  
Vol 3 (6) ◽  
pp. 1653-1663 ◽  
Author(s):  
Sherif Ganem ◽  
Shun-Wen Lu ◽  
Bee-Na Lee ◽  
David Yu-Te Chou ◽  
Ruthi Hadar ◽  
...  
Keyword(s):  
Map Kinase ◽  
G Protein ◽  
Signal Transduction Pathway ◽  
Cochliobolus Heterostrophus ◽  
Wild Type ◽  
Heterotrimeric G Protein ◽  
Reproductive Ability ◽  
Pathway Gene ◽  
Mitogen Activated Protein ◽  
Nuclear Degradation

ABSTRACT Previous work established that mutations in mitogen-activated protein (MAP) kinase (CHK1) and heterotrimeric G-protein α (Gα) subunit (CGA1) genes affect the development of several stages of the life cycle of the maize pathogen Cochliobolus heterostrophus. The effects of mutating a third signal transduction pathway gene, CGB1, encoding the Gβ subunit, are reported here. CGB1 is the sole Gβ subunit-encoding gene in the genome of this organism. cgb1 mutants are nearly wild type in vegetative growth rate; however, Cgb1 is required for appressorium formation, female fertility, conidiation, regulation of hyphal pigmentation, and wild-type virulence on maize. Young hyphae of cgb1 mutants grow in a straight path, in contrast to those of the wild type, which grow in a wavy pattern. Some of the phenotypes conferred by mutations in CGA1 are found in cgb1 mutants, suggesting that Cgb1 functions in a heterotrimeric G protein; however, there are also differences. In contrast to the deletion of CGA1, the loss of CGB1 is not lethal for ascospores, evidence that there is a Gβ subunit-independent signaling role for Cga1 in mating. Furthermore, not all of the phenotypes conferred by mutations in the MAP kinase CHK1 gene are found in cgb1 mutants, implying that the Gβ heterodimer is not the only conduit for signals to the MAP kinase CHK1 module. The additional phenotypes of cgb1 mutants, including severe loss of virulence on maize and of the ability to produce conidia, are consistent with CGB1 being unique in the genome. Fluorescent DNA staining showed that there is often nuclear degradation in mature hyphae of cgb1 mutants, while comparable wild-type cells have intact nuclei. These data may be genetic evidence for a novel cell death-related function of the Gβ subunit in filamentous fungi.

The yeast G-protein homolog is involved in the mating pheromone signal transduction system

1989 ◽  
Vol 9 (1) ◽  
pp. 152-158
Author(s):  
H A Fujimura
Keyword(s):  
Signal Transduction ◽  
G Protein ◽  
Signal Transduction Pathway ◽  
Alpha Cells ◽  
Mating Pheromone ◽  
Gene Encoding ◽  
Pheromone Response Pathway ◽  
New Type ◽  
Sterile Mutant ◽  
Guanine Nucleotide Binding

I have isolated a new type of sterile mutant of Saccharomyces cerevisiae, carrying a single mutant allele, designated dac1, which was mapped near the centromere on chromosome VIII. The dac1 mutation caused specific defects in the pheromone responsiveness of both a and alpha cells and did not seem to be associated with any pleiotropic phenotypes. Thus, in contrast to the ste4, ste5, ste7, ste11, and ste12 mutations, the dac1 mutation had no significant effect on such constitutive functions of haploid cells as pheromone production and alpha-factor destruction. The characteristics of this phenotype suggest that the DAC1 gene encodes a component of the pheromone response pathway common to both a and alpha cells. Introduction of the GPA1 gene encoding an S. cerevisiae homolog of the alpha subunit of mammalian guanine nucleotide-binding regulatory proteins (G proteins) into sterile dac1 mutants resulted in restoration of pheromone responsiveness and mating competence to both a and alpha cells. These results suggest that the dac1 mutation is an allele of the GPA1 gene and thus provide genetic evidence that the yeast G protein homolog is directly involved in the mating pheromone signal transduction pathway.

Stoichiometry of G protein subunits affects the Saccharomyces cerevisiae mating pheromone signal transduction pathway

1990 ◽  
Vol 10 (2) ◽  
pp. 510-517
Author(s):  
G M Cole ◽  
D E Stone ◽  
S I Reed
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Signal Transduction ◽  
G Protein ◽  
Signal Transduction Pathway ◽  
Mating Pheromone ◽  
Gal1 Promoter ◽  
Gamma Subunits ◽  
Adaptation Response ◽  
High Level

The Saccharomyces cerevisiae GPA1, STE4, and STE18 genes encode products homologous to mammalian G-protein alpha, beta, and gamma subunits, respectively. All three genes function in the transduction of the signal generated by mating pheromone in haploid cells. To characterize more completely the role of these genes in mating, we have conditionally overexpressed GPA1, STE4, and STE18, using the galactose-inducible GAL1 promoter. Overexpression of STE4 alone, or STE4 together with STE18, generated a response in haploid cells suggestive of pheromone signal transduction: arrest in G1 of the cell cycle, formation of cellular projections, and induction of the pheromone-inducible transcript FUS1 25- to 70-fold. High-level STE18 expression alone had none of these effects, nor did overexpression of STE4 in a MATa/alpha diploid. However, STE18 was essential for the response, since overexpression of STE4 was unable to activate a response in a ste18 null strain. GPA1 hyperexpression suppressed the phenotype of STE4 overexpression. In addition, cells that overexpressed GPA1 were more resistant to pheromone and recovered more quickly from pheromone than did wild-type cells, which suggests that GPA1 may function in an adaptation response to pheromone.

Effects of calcium on membrane potential and sodium influx in barnacle muscle fibers

1983 ◽  
Vol 244 (3) ◽  
pp. C297-C302 ◽  
Author(s):  
S. S. Sheu ◽  
M. P. Blaustein
Keyword(s):  
Membrane Potential ◽  
Muscle Fibers ◽  
Cation Channels ◽  
Channel Blockers ◽  
Sodium Influx ◽  
Permeable Channel ◽  
Barnacle Muscle ◽  
Nonselective Cation Channels ◽  
Flux Measurements ◽  
Barnacle Muscle Fibers

The influence of internal and external Ca2+ on membrane potential and 22Na influx were tested in internally perfused giant barnacle muscle fibers. The fibers depolarized by about 2-3 mV, and Na+ influx increased when external Ca2+ was removed. These effects were inhibited and reversed by adding 2 mM La3+ externally but not by tetrodotoxin (TTX). Ca2+ channel blockers did not prevent the depolarization. Increasing internal free Ca2+ ([Ca2+]i) from 10(-7) to 10(-5) M also stimulated Na+ influx and depolarized the fibers by a few millivolts. Neither external La3+ nor TTX prevented the effects of raising [Ca2+]i; however, internal tetrabutylammonium ions depolarized the fibers and attenuated the internal Ca2+-dependent effects. These data are consistent with the idea that removal of external Ca2+ activates a La3+-sensitive channel that is permeable to Na+; raising [Ca2+]i activates a La2+-insensitive, Na+-permeable channel that may be similar to the internal Ca2+-activated nonselective cation channels observed in cardiac muscle. The results demonstrate that all Na+ (and Ca2+) fluxes that do not contribute to Na-Ca exchange must be carefully identified before the exchange stoichiometry can be determined from Na+ and Ca2+ flux measurements.

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