Physiology and biophysics of chloride and cation cotransport across cell membranes

Many important questions remain to be answered about the mechanism that mediates coupled Na,K,Cl cotransport. We still do not know what the ATP requirement involves. Is ATP the direct energy source? Such an energy source does not seem to be necessary, inasmuch as the net free energy in the combined transmembrane chemical gradients of Na, K, and Cl is quite sufficient to maintain the observed high Cl(i). Could a protein kinase-mediated mechanism be responsible for the ATP requirement? How does reducing Cl(i) stimulate the transporter? What are the kinetic relationships for the co-ions at the outward- and inward-facing transport sites? Are they symmetrical? Can the squid axon regulate its cell volume? If so, is the Na,K,Cl transporter directly involved? Thus, the squid axon remains a fruitful preparation to study a transport mechanism similar to that found in a variety of cells. Its large size confers unique experimental advantages that should help us in our quest to understand this widely distributed transport mechanism.     

Kinetic comparison of ouabain-resistant K:CI fluxes (K:Cl [Co]-transport) stimulated in sheep erythrocytes by membrane thiol oxidation and alkylation

Summary The stimulatory effects of two thiol (SH) group oxidants, methylmethane thiosulfonate (MMTS) and diazene dicarboxylic acid bis [N,N-dimethylamide] (diamide), on the kinetics of ouabain-resistant (OR) K:Cl [co]-transport in low K (LK) sheep red blood cells were compared with the effects of alkylating agents, notably N-ethylmaleimide (NEM). At low concentrations, both MMTS and diamide stimulated K:CI [co]-transportv and with a latency period, as measured by OR zero-trans K efflux and OR uptake of external Rb, Rb_o, as K congener in Cl and NO₃ media. At high concentrations the effect of diamide saturated, and that of MMTS disappeared. The stimulatory effect of MMTS was partially reversed by the reducing agent dithiothreitol (DTT) known to fully restore the diamide-activated K flux (Lauf, J. Memb. Biol. 101:179–188, 1988). In diamide pre-equilibrated LK sheep red cells, the K_m of K:Cl [co]-transport for external Cl, Cl_o, was 84.3 mM, and 18.7 mM for Rb_o, with nearly identical V_max values around 4 mmol Rb/L cells × h for K (Rb) fluxes in Cl and after correction for the small Cl-independent component. Zero net K (Rb) flux existed at K_c (cell K)/Rb_o concentration ratios, [K]_c/[Rb]_c, of 0.8 i.e. when the electrochemical driving forces across the membrane were about equal. The measured K efflux/Rb influx ratios were almost twice those predicted from [K]_c/[Rb]_o and the Cl equilibrium potential suggesting that the diamide-stimulated K (Rb) flux may occur through non-diffusional, carrier-mediated transport. The effects of NEM and of A23187 plus/minus Ca or chelators on K: [co]Cl-transport (Lauf, Am. J. Physiol. 249:C271–278, 1985) consisted primarily of V_max changes. Thus, all chemical interventions resulted in an increase of the number of actively transporting K:Cl [co]-transport units or an augmented turnover number per existing site.     

Ion transport in a human lens epithelial cell line exposed to hyposmotic and apoptotic stress

Membrane transport changes in human lens epithelial (HLE‐B3) cells under hyposmotic and apoptotic stress were compared. Cell potassium content, K_i, uptake of the K congener rubidium, Rb_i, and water content were measured after hyposmotic stress induced by hypotonicity, and apoptotic stress by the protein‐kinase inhibitor staurosporine (STP). Cell water increased in hyposmotic (150 mOsm) as compared to isosmotic (300 mOsm) balanced salt solution (BSS) by >2‐fold at 5 min and decreased within 15 min to baseline values accompanied by a 40% K_i loss commensurate with cell swelling and subsequent cell shrinkage likely due to regulatory volume decrease (RVD). Loss of K_i, and accompanying water, and Rb_i uptake in hyposmotic BSS were prevented by clotrimazole (CTZ) suggesting water shifts associated with K and Rb flux via intermediate conductance K (IK) channels, also detected at the mRNA and protein level. In contrast, 2 h after 2 µM STP exposure, the cells lost ～40% water and ～60% K_i, respectively, consistent with apoptotic volume decrease (AVD). Indeed, water and K_i loss was at least fivefold greater after hyposmotic than after apoptotic stress. High extracellular K and 2 mM 4‐aminopyridine (4‐AP) but not CTZ significantly reduced apoptosis. Annexin labeling phosphatidylserine (PS) at 15 min suggested loss of lipid asymmetry. Quantitative PCR revealed significant IK channel expression during prolonged hyposmotic stress. Results suggest in HLE‐B3 cells, IK channels likely partook in and were down regulated after RVD, whereas pro‐apoptotic STP‐activation of 4‐AP‐sensitive voltage‐gated K channels preceded or accompanied PS externalization before subsequent apoptosis. J. Cell. Physiol. 223: 110–122, 2010. © 2009 Wiley‐Liss, Inc.