Srisaila Basavappa

Regulation of volume‐activated chloride channels by P‐glycoprotein: phosphorylation has the final say!

1 P‐glycoprotein (Pgp) is a transmembrane transporter causing efflux of a number of chemically unrelated drugs and is responsible for resistance to a variety of anticancer drugs during chemotherapy. 2 Pgp overexpression in cells is also associated with volume‐activated chloride channel activity; Pgp is thought to regulate such activity. 3 Reversible phosphorylation is a possible mechanism for regulating the transport and chloride channel regulation functions of Pgp. Protein kinase C (PKC) is a good candidate for inducing such phosphorylation. 4 Hierarchical multiple phosphorylation (e.g. of different serines and with different PKC isoforms) may shuttle the protein between its different states of activity (transport or channel regulation). Cell volume changes may trigger phosphorylation of Pgp at sites causing inhibition of transport. 5 The possible regulation of chloride channels by Pgp and the potential involvement of reversible phosphorylation in such regulation is reviewed.

GTP-binding proteins regulate high conductance anion channels in rat bile duct epithelial cells.

SummaryEpithelial cells from the intrahepatic bile duct contribute to bile formation, but little is known of the cellular mechanisms responsible. In these studies, we have characterized the endogenous GTP-binding proteins (G proteins) present in these cells and evaluated their role in regulation of high conductance anion channels. G proteins were identified in purified plasma membranes of isolated bile duct epithelial cells using specific antisera on Western blots, and ion channel activity was measured in excised inside-out membrane patches using patch-clamp recording techniques. In patches without spontaneous channel activity, addition of cholera toxin to the cytoplasmic surface had no effect (n=10). Addition of pertussis toxin caused an activation of channels in 13/34 (38%) attempts, as detected by an increase in channel open probability. Activated channels were anion selective (gluconate/Cl− permeability ratio of 0.17±0.04) and had a unitary conductance of ∼380 pS. Channel open probability was also increased by the nonhydrolyzable GDP analogue guanosine 5′-0-(2-thiodiphosphate) in 8/14 (57%) attempts. In contrast, channel open probability was rapidly and reversibly decreased by the nonhydrolyzable analogue of GTP 5′guanylylimidodiphosphate in 7/9 (78%) attempts. Western blotting with specific antisera revealed that both Giα−2 and Giα−3 were present in significant amounts, whereas Giα−1 and Goα were not detected. These studies indicate that in bile duct epithelial cells, high conductance anion channels are inhibited, in a membrane-delimited manner, by PTX-sensitive G proteins.

Effects of calcium, calmodulin, protein kinase C and protein tyrosine kinases on volume‐activated taurine efflux in human erythroleukemia cells

The effects of calcium, calmodulin, protein kinase C (PKC) and protein tyrosine kinase (PTK) modulators were examined on the volume‐activated taurine efflux in the erythroleukemia cell line K562. Exposure to hypoosmotic solution significantly increased taurine efflux and intracellular calcium concentration ([Ca2+]i). The Ca2+ channel blockers La3+ (1 mM), verapamil (200 μM) and nifedipine (100 μM) inhibited the hypoosmotically‐induced [Ca2+]i increase by more than 90%, while the volume‐activated taurine efflux was inhibited by 61.3 ± 9.5, 74.1 ± 9.3 and 38.0 ± 1.5%, respectively. Furthermore, the calmodulin inhibitors W7 (50 μM) and trifluoperazine (10 μM) and the Ca2+/calmodulin‐dependent protein kinase II inhibitor KN‐62 (2 μM) significantly blocked the volume‐activated taurine efflux by 93.4 ± 2.7, 77.9 ± 3.5 and 61.3 ± 15.8%, respectively. In contrast, the PKC inhibitor staurosporine (200 nM) or the PKC activator phorbol 12‐myristate 13‐acetate (100 nM) did not have significant effects on the volume‐activated taurine efflux. However, pretreatment with PTK inhibitors genistein, tyrphostin A25, and tyrphostin A47 blocked the volume‐activated taurine efflux. These results suggest that the volume‐activated taurine efflux in K562 cells may not directly involve Ca2+, but may require the presence of calmodulin and/or PTK.

Calcium-dependent, swelling-activated K+ conductance in human neuroblastoma cells

In most mammalian cells, regulatory volume decrease (RVD) is mediated by swelling-activated Cl(-) and K(+) channels. Previous studies in the human neuroblastoma cell line CHP-100 have demonstrated that exposure to hypoosmotic solutions activates Cl(-) channels which are sensitive to Ca(2+). Whether a Ca(2+)-dependent K(+) conductance is activated after cell swelling was investigated in the present studies. Reducing the extracellular osmolarity from 290 to 190 mOsm/kg H(2)O rapidly activated 86Rb effluxes. Hypoosmotic stress also increased cytosolic Ca(2+) in fura-2 loaded cells. Pretreatment with 2.5 mM EGTA and nominally Ca(2+) free extracellular solution significantly decreased the hypoosmotically induced rise in cytosolic Ca(2+) and the swelling-activated 86Rb efflux. In cell-attached patch-clamp studies, decreasing the extracellular osmolarity activated a K(+) conductance that was blocked by Ba(2+). In addition, the swelling-activated K(+) channels were significantly inhibited in the presence of nominally free extracellular Ca(2+) and 2.5mM EGTA. These results suggest that in response to hypoosmotic stress, a Ca(2+)-dependent K(+) conductance is activated in the human neuroblastoma cell line CHP-100.

cAMP-regulated chloride currents in CHO cells.

We examined whether elevations in cAMP levels increase membrane chloride permeability in native CHO cells by measuring whole cell chloride currents and efflux of 125I and 36Cl. With 20 microM forskolin, no significant effect was seen on whole cell currents. However, 100 microM forskolin increased both whole cell chloride currents and the rate of 125I and 36Cl efflux. Forskolin-activated currents showed a linear current-voltage relationship in solutions with symmetrical chloride concentrations and reversal potential changed in the direction anticipated for a chloride-selective current when chloride was replaced with gluconate. These results indicate that native CHO cells exhibit cAMP-regulated chloride conductance pathways which become apparent only after large elevations in intracellular cAMP levels.