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Fish Physiology and Biochemistry

, Volume 44, Issue 2, pp 583–597 | Cite as

Biochemical properties of the sensitivity to GABAAergic ligands, Cl/HCO3 -ATPase isolated from fish (Cyprinus carpio) olfactory mucosa and brain

  • Sergey Menzikov
Article

Abstract

This paper presents a comparative study of the roles of Cl and HCO3 in the functioning of the GABAAR-associated Cl/HCO3 -ATPase of the plasma membranes of the olfactory sensory neurons (OSNs) and mature brain neurons (MBNs) of fish. The ATPase activity of OSNs and its dephosphorylation were increased twofold by Cl(15–30 mmol l−1), whereas the enzyme from MBNs was not significantly affected by Cl. By contrast, HCO3 (15–30 mmol l−1) significantly activated the MBN enzyme and its dephosphorylation, but had no effect on the OSN ATPase. The maximum ATPase activity and protein dephosphorylation was observed in the presence of both Cl(15 mmol l−1)/HCO3 (27 mmol l−1) and these activities were inhibited in the presence of picrotoxin (100 μmol l−1), bumetanide (150 μmol l−1), and DIDS (1000 μmol l−1). SDS-PAGE revealed that ATPases purified from the neuronal membrane have a subunit with molecular mass of ~ 56 kDa that binds [3H]muscimol and [3H]flunitrazepam. Direct phosphorylation of the enzymes in the presence of ATP-γ-32P and Mg2+, as well as Cl/HCO3 sensitive dephosphorylation, is also associated with this 56 kDa peptide. Both preparations also showed one subunit with molecular mass 56 kDa that was immunoreactive with GABAAR β3 subunit. The use of a fluorescent dye for Cl demonstrated that HCO3 (27 mmol l−1) causes a twofold increase in Cl influx into proteoliposomes containing reconstituted ATPases from MBNs, but HCO3 had no effect on the reconstituted enzyme from OSNs. These data are the first to demonstrate a differential effect of Cl and HCO3 in the regulation of the Cl/HCO3 -ATPases functioning in neurons with different specializations.

Keywords

Fish Brain Olfactory mucosa Cl/HCO3-ATPase Phosphorylation Transport 

Abbreviations

OSNs

olfactory sensory neurons

BNs

brain neurons

MBNs

mature brain neurons

IMNs

immature neuron

GABAAR

GABAA receptor

EM

membrane potential

ECl

equilibrium potential for Cl

[Cl]i

the intracellular concentration of Cl

CССs

cation-chloride co-transporters

KCCs

K+/Cl co-transporters

NKCCs

Na+/K+/2Cl co-transporters

AEs

anion exchangers

Picr

picrotoxin

Bum

bumetanide

Fur

furosemide

DIDS

Disodium 4,4′-diisothiocyanatostilbene-2,2′-disulfonate

VO42−

o-vanadate

MOPS

3-(N-morpholino)propanesulfonic acid

PVDF

polyvinylidene difluoride (PVDF) membranes

TBST

tris-buffered saline

Notes

Compliance with ethical standards

The experimental investigations of the material was approved by the Ethical Committee of FSBSI “Institute of general pathology and pathophysiology” (No 01-01/147 from October 12, 2009) and performed according to the principles expressed in the Declaration of Helsinki revised by WMA, Fortaleza, Brazil, 2013.

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Authors and Affiliations

  1. 1.Department Russian Academy of ScienceInstitute of General Pathology and PathophysiologyMoscowRussia

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