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CAMP/protein kinase A activates cystic fibrosis transmembrane conductance regulator for ATP release from rat skeletal muscle during low pH or contractions.

ABSTRACT: We have shown that cystic fibrosis transmembrane conductance regulator (CFTR) is involved in ATP release from skeletal muscle at low pH. These experiments investigate the signal transduction mechanism linking pH depression to CFTR activation and ATP release, and evaluate whether CFTR is involved in ATP release from contracting muscle. Lactic acid treatment elevated interstitial ATP of buffer-perfused muscle and extracellular ATP of L6 myocytes: this ATP release was abolished by the non-specific CFTR inhibitor, glibenclamide, or the specific CFTR inhibitor, CFTR(inh)-172, suggesting that CFTR was involved, and by inhibition of lactic acid entry to cells, indicating that intracellular pH depression was required. Muscle contractions significantly elevated interstitial ATP, but CFTR(inh)-172 abolished the increase. The cAMP/PKA pathway was involved in the signal transduction pathway for CFTR-regulated ATP release from muscle: forskolin increased CFTR phosphorylation and stimulated ATP release from muscle or myocytes; lactic acid increased intracellular cAMP, pCREB and PKA activity, whereas IBMX enhanced ATP release from myocytes. Inhibition of PKA with KT5720 abolished lactic-acid- or contraction-induced ATP release from muscle. Inhibition of either the Na(+)/H(+)-exchanger (NHE) with amiloride or the Na(+)/Ca(2+)-exchanger (NCX) with SN6 or KB-R7943 abolished lactic-acid- or contraction-induced release of ATP from muscle, suggesting that these exchange proteins may be involved in the activation of CFTR. Our data suggest that CFTR-regulated release contributes to ATP release from contracting muscle in vivo, and that cAMP and PKA are involved in the activation of CFTR during muscle contractions or acidosis; NHE and NCX may be involved in the signal transduction pathway.

SUBMITTER: Tu J

PROVIDER: S-EPMC3511434 | biostudies-literature | 2012

REPOSITORIES: biostudies-literature

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cAMP/protein kinase A activates cystic fibrosis transmembrane conductance regulator for ATP release from rat skeletal muscle during low pH or contractions.

Tu Jie J Lu Lin L Cai Weisong W Ballard Heather J HJ

PloS one 20121130 11

We have shown that cystic fibrosis transmembrane conductance regulator (CFTR) is involved in ATP release from skeletal muscle at low pH. These experiments investigate the signal transduction mechanism linking pH depression to CFTR activation and ATP release, and evaluate whether CFTR is involved in ATP release from contracting muscle. Lactic acid treatment elevated interstitial ATP of buffer-perfused muscle and extracellular ATP of L6 myocytes: this ATP release was abolished by the non-specific ...[more]

PMID: 23226244

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Project description:Background: Most cystic fibrosis is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene that lead to protein misfolding and degradation by the ubiquitin-proteasome system. Previous studies demonstrated that PIAS4 facilitates the modification of wild-type (WT) and F508del CFTR by small ubiquitin-like modifier (SUMO)-1, enhancing CFTR biogenesis by slowing immature CFTR degradation and producing increased immature CFTR band B. Methods: We evaluated two correction strategies using misfolding mutants, including the common variant, F508del. We examined the effects on mutant expression of co-expression with PIAS4 (E3 SUMO ligase), and/or the corrector, C18. To study the impact of these correction conditions, we transfected CFBE410- cells, a bronchial epithelial cell line, with a CFTR mutant plus: (1) empty vector, (2) empty vector plus overnight 5 μM C18, (3) PIAS4, and (4) PIAS4 plus C18. We assessed expression at steady state by immunoblot of CFTR band B, and if present, band C, and the corresponding C:B band ratio. The large PIAS4-induced increase in band B expression allowed us to ask whether C18 could act on the now abundant immature protein to enhance correction above the control level, as reported by the C:B ratio. Results: The data fell into three mutant CFTR categories as follows: (1) intransigent: no observable band C under any condition (i.e., C:B = 0); (2) throughput responsive: a C:B ratio less than control, but suggesting that the increased band C resulted from PIAS4-induced increases in band B production; and (3) folding responsive: a C:B ratio greater than control, reflecting C18-induced folding greater than that expected from increased throughput due to the PIAS4-induced band B level. Conclusion: These results suggest that the immature forms of CFTR folding intermediates occupy different loci within the energetic/kinetic folding landscape of CFTR. The evaluation of their properties could assist in the development of correctors that can target the more difficult-to-fold mutant conformations that occupy different sites within the CFTR folding pathway.

Dataset Information

CAMP/protein kinase A activates cystic fibrosis transmembrane conductance regulator for ATP release from rat skeletal muscle during low pH or contractions.

Publications

cAMP/protein kinase A activates cystic fibrosis transmembrane conductance regulator for ATP release from rat skeletal muscle during low pH or contractions.

Similar Datasets

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