Pw. Flatman et al., ROLE OF PROTEIN-KINASES IN REGULATING SHEEP ERYTHROCYTE K-CL COTRANSPORT, American journal of physiology. Cell physiology, 40(1), 1996, pp. 255-263
K-Cl cotransport in sheep erythrocytes can be activated by treatment e
ither with A-23187 and EDTA to reduce concentration of internal ionize
d Mg ([Mg](i)) to submicromolar levels, with staurosporine, a potent k
inase inhibitor, or with N-ethylmaleimide (NEM). Activation by these m
aneuvers is prevented and reversed by genistein [inhibition constant (
K-i) of 15 mu M], which inhibits tyrosine kinases (TK). The related gl
ycosidated compound genistin, which does not inhibit TK, does not inhi
bit transport, whereas another TK inhibitor, tyrphostin B46, inhibits
both basal and stimulated transport (K-i of 28 mu M). Cotransport acti
vation by NEM is prevented and reversed by the phosphatase inhibitor,
calyculin A, and activation by staurosporine occurs only if cells cont
ain ATP. Increasing [Mg](i) inhibits cotransport in the presence of ca
lyculin A whether or not staurosporine is present as well. Our work su
ggests that genistein inhibits cotransport through a TK and that staur
osporine and NEM activate cotransport, probably through inhibition of
other kinases, causing stimulation through dephosphorylation of a prot
ein (possibly the transporter itself) by a serine/threonine phosphatas
e. [Mg](i) inhibits cotransport by activating a kinase (concentration
for half-maximal activation of 10 mu M) that phosphorylates this prote
in.