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  • The identity of the K transport pathway activated by NEM


    The identity of the K+ transport pathway activated by NEM remains unclear. The pathway does not require the presence of Gardos channel KCNN4 or K–Cl cotransporters KCC3 and KCC1, but like these pathways is sensitive to the presence of hemoglobin S or other sickling hemoglobins. Although partially sensitive to chloroquine and amiloride, the lack of dependence on temperature or extracellular pH and the inability to test for phloretin-sensitivity fail to provide additional support for K+(Na+)/H+ exchange as the NEM-stimulated Cl−-independent K+ efflux pathway. The Gardos channel has been reported to be NEM-insensitive [37], but NEM has multiple effects on human erythrocytes (beyond that on K–Cl cotransport) that suggest consideration as possible modifiers or regulators of Cl−-independent K+ efflux in mouse erythrocytes. Among the human erythrocyte transport activities inhibited by mM NEM are 45Ca uptake induced by plasmalemmal Ca-ATPase inhibitor vanadate [48], the nonselective voltage-dependent cation conductance [49], sulfate transport [50], low affinity cationic amino A 804598 transport (system y+L) [51], and equilibrative nucleobase transport [52]. Multiple transport systems may be indirectly modulated by NEM's promotion of the tetramer-to-dimer transition of spectrin, attributed to weakened interaction of ankyrin with AE1/band 3 [53]; by NEM's activation of phospholipid scramblase, inhibition of aminophospholipid translocase (flippase), and the NEM-associated reduced Ca requirement for phospholipid scrambling in both mouse and human erythrocytes [54]; by NEM's inhibition of erythroid pertussis toxin-sensitive G proteins [55], [56], [57]; by NEM's inhibition of serine phosphatases PP1 and PP2A [58]; by NEM's enhanced plasma membrane translocation of calpain-1 [59] and of tyrosine phosphatase SHP1 (independent of Band 3 tyrosine phosphorylation) [60]; and by NEM's inhibition of calpain at multi-mM concentrations [61]. The Cl−-free conditions required for detection of NEM-stimulated Cl−-independent K+ flux may also activate WNK1 kinase by depletion of intracellular Cl− [62], with consequences to multiple transport systems in addition to the K-Cl cotransporters. Sulfhydryl group redox state also regulates several pertinent transport activities not yet known to be present in circulating erythrocytes. Among these, NEM activates the isothiocyanate-activated cation channel TRPA1 [63] and the voltage-gated K+ channels (M channels) Kv7.2, Kv7.4, and Kv7.5 [64] (also activated by H2O2 in a dithiothreitol-reversible manner [65]). In summary, we have characterized an NEM-activated Cl−-independent K+ transport pathway previously noted in mouse erythrocytes but not further investigated at that time. This pathway is independent of the Gardos channel KCNN4 and the K–Cl cotransporters KCC3 and KCC1, and exhibits lower NEM-sensitivity than that of mouse erythroid K–Cl cotransport. The presence of HbSAD increases both the NEM-sensitivity and the magnitude of stimulation of the Cl−-independent K+ efflux pathway. The pathway is inhibited by mM concentrations of chloroquine and amiloride, but does not share several other properties of K+(Na+)/H+ exchange as described in human erythrocytes. This K+ efflux pathway is also partially inhibited by Ba. Additional studies will be needed to establish the functional and molecular identities of the protein(s) that mediate NEM-stimulated Cl−-independent K+ efflux of the mouse erythrocyte, and to learn whether the reported corresponding activity of human erythrocytes might be orthologous. Erythrocytes of the triple knockout mouse will be useful in maximally isolating NEM-stimulated Cl−-independent K+ efflux for further study, and in characterizing its regulation by HbSAD or other sickling hemoglobins in oxygenated and deoxygenated states.
    Role of the Funding Source
    Conflict of Interest
    Acknowledgments This work was supported by NIH grants HL090632 (AR) and HL077655 (SLA). We thank Edward S. Kim and Katherine K. Nishimura (Beth Israel Deaconess Medical Center) for their technical assistance. We also thank James E. Melvin (National Institute of Dental and Craniofacial Research) and Thomas J. Jentsch (Leibniz-Institut fur Molekulare Pharmakologie and Max-Delbruck-Centrum fur Molekulare Medizin, Berlin) for genetically modified mice.