AJP - Cell Physiology, Vol 267, Issue 4 C1002-C1012, Copyright © 1994 by American Physiological Society
Labeling of the Amphiuma erythrocyte K+/H+ exchanger with H2DIDS
H. M. Maldonado and P. M. Cala
Department of Human Physiology, University of California at Davis, School of Medicine 95616.
Subsequent to swelling, the Amphiuma red blood cells lose K+, Cl-, and
water until normal cell volume is restored. Net solute loss is the result
of K+/H+ and Cl-/HCO3- exchangers functionally coupled through changes in
pH and therefore HCO3-. Whereas the Cl-/HCO3- exchanger is constitutively
active, K+/H+ actively is induced by cell swelling. The constitutive
Cl-/HCO3- exchanger is inhibited by low concentrations (< 1 microM) of
4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) or H2DIDS, yet the
concentration of H2DIDS > 25 microM irreversibly modifies the K+/H+
exchanger in swollen cells. We exploited the volume-dependent irreversible
low-affinity reaction between H2DIDS and the K+/H+ to identify the
protein(s) associated with K+/H+ exchange activity. Labeling of the
membrane proteins of intact cells with 3H2DIDS results in high-affinity
labeling of a broad 100-kDa band, thought to be the anion exchanger.
Additional swelling-dependent low-affinity labeling at 110 kDa suggests the
possibility of a volume-induced population of anion exchangers. Finally,
the correlation between volume-sensitive K+/H+ modification and
low-affinity labeling suggests that transport activity is associated with a
protein of approximately 85 kDa. Although a 55-kDa protein is also labeled,
it is a less likely candidate, since label incorporation and transport
modification are less well correlated than that of the 85- and 110-kDa
proteins.
