Stimulation of human erythrocyte K-Cl cotransport and protein phosphatase type 2A by n-ethylmaleimide: Role of intracellular Mg++

Bize I., Guvenc B., Buchbinder G., Brugnara C.

JOURNAL OF MEMBRANE BIOLOGY, vol.177, no.2, pp.159-168, 2000 (SCI-Expanded) identifier identifier identifier

  • Publication Type: Article / Article
  • Volume: 177 Issue: 2
  • Publication Date: 2000
  • Doi Number: 10.1007/s002320001109
  • Journal Indexes: Science Citation Index Expanded (SCI-EXPANDED), Scopus
  • Page Numbers: pp.159-168
  • Çukurova University Affiliated: Yes


An increase in the activity of membrane-associated protein phosphatase type 1 (mb-PP1) is associated with stimulation of erythrocyte K-Cl cotransport (KCC). We have recently proposed that membrane-associated protein phosphatase type 2A (mb-PP2A) is also involved in KCC regulation by cell swelling (Bize et al., 1999. Am. J. Physiol. 277:C899-C912). We used two protein phosphatase inhibitors, okadaic acid (OA) and calyculin A (CalA), and two KCC activating treatments, n-ethylmaleimide (NEM) and Mg-i(++)-depletion, and determined KCC transport activity and mb-PPI and mb-PP2A activities. OA, an inhibitor of erythrocyte mb-PP2A, partially prevents stimulation of KCC activity by NEM but not by Mg-i(++)-depletion. CalA, an inhibitor of both mb-PP1 and mb-PP2A prevents stimulation of KCC activity by both treatments. NEM and Mg-i(++)-depletion inhibit mb-PP1 activity, suggesting that activation of KCC can take place in the absence of mb-PP1 activation. Mb-PP2A activity is stimulated in NEM-treated cells but not in Mg-i(++)-depleted cells. In NEM-treated cells, Mg-i(++)-depletion inhibits both KCC and mb-PP2A. In Mg-i(++)-depleted cells, NEM does not stimulate KCC or mb-PP2A. The strong correlation between KCC stimulation and mb-PP2A stimulation provides further support to the idea that mb-PP2A plays an important role in KCC regulation. Our results are consistent with the hypothesis that KCC regulation involves at least two distinguishable phosphorylation sites.