Stimulation of human erythrocyte K-Cl cotransport and protein phosphatase type 2A by n-ethylmaleimide: role of intracellular Mg++.
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-PP1 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.
Bize
I
I
Department of Pathology, The Children's Hospital, 300 Longwood Ave, Farley 715, Boston, MA 02115, USA.
Güvenç
B
B
Buchbinder
G
G
Brugnara
C
C
D002352:Carrier Proteins / Q000097:blood*; D002712:Chlorides / Q000097:blood*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004910:Erythrocyte Membrane / Q000187:drug effects / Q000378:metabolism*; D005033:Ethylmaleimide / Q000494:pharmacology*; D006801:Humans; D066298:In Vitro Techniques; D008274:Magnesium / Q000097:blood*; D019319:Okadaic Acid / Q000494:pharmacology; D010749:Phosphoprotein Phosphatases / Q000097:blood*; D011188:Potassium / Q000097:blood*; D027981:Symporters; D000096922:K Cl- Cotransporters
D002352:Carrier Proteins; D002712:Chlorides; D004791:Enzyme Inhibitors; D005033:Ethylmaleimide; D008274:Magnesium; D019319:Okadaic Acid; D010749:Phosphoprotein Phosphatases; D011188:Potassium; D027981:Symporters; D000096922:K Cl- Cotransporters