Abstract
The regulatory volume decrease (RVD) of a renal cortical collecting duct cell line (RCCT-28A) exposed to a hypotonic solution was studied using electronic cell sizing to measure cell volume and the patch clamp technique to measure Cl- channel activity. Results demonstrate that RVD was mediated in part by KCl loss through separate K+ and Cl- channels. The Cl- channel had a conductance of 305 pS and was activated by cell swelling, membrane stretch, and disruption of F-actin by dihydrocytochalasins. In contrast, stabilizing F-actin with phalloidin prevented swelling and stretch activation of the Cl- channel and inhibited the RVD. Thus, the state of actin polymerization regulates the probability of the 305 pS Cl- channel being open. Short actin filaments activate whereas long actin filaments inactivate the channel. Taken together, our studies suggest that RVD in this renal collecting duct cell line cell is mediated in part by a 305 pS Cl- channel, which is activated, during cell swelling, by a signaling pathway that includes disruption of F-actin.
Highlights
Actin-based Cytoskeleton Regulates a Chloride Channel anCdell Volume in a Renal Cortical Collecting Duct Cell Line*
Measurement of Cell VoZume-Cell volume was measured by elec- Characterization of RVD-The first series of experiments tronic cell sizing a s described in detailpreviously (26-28).Briefly, cells were conducted to determine if RCCT-28A cells volume reguwere grown on tissue culture-treated polystyrene flasks and were har- late inresponse to a hypotonic solution and, if so, to determine vested by a mild trypsinization protocol (0.05% trypsin and 0.53 m~ EDTA in a 0 Ca2+ and0 Mg2+phosphate-buffered saline solution). 8 x lo6 cells were suspendedin 4 ml of an isotonic cell solutionor 60min
Channels thatwere activated during con- andor fragment F-actin (39-43) in inside-out patches of the version from the cell-attached to the inside-out configuration apical membrane.A representative experiment illustrating the were more sensitive to small increases in negative pressure effects of dihydrocytochalasin B (DHCB) on a C1- channel is than channels thawt ere not activated duringformation of the shown in Fig. 6.DHCB increased thePoof quiescent and exciinside-out configuration
Summary
Measurement of Cell VoZume-Cell volume was measured by elec- Characterization of RVD-The first series of experiments tronic cell sizing a s described in detailpreviously (26-28).Briefly, cells were conducted to determine if RCCT-28A cells volume reguwere grown on tissue culture-treated polystyrene flasks and were har- late inresponse to a hypotonic solution and, if so, to determine vested by a mild trypsinization protocol (0.05% trypsin and 0.53 m~ EDTA in a 0 Ca2+ and0 Mg2+phosphate-buffered saline solution). 8 x lo cells were suspendedin 4 ml of an isotonic cell solution (inm:, NaCl; 2.4,KCl; 0.6,MgC12; 1.2,CaC12; 0.6,KH2P04; 7.5,Hepes; 10, glucose; 160,sucrose; pH7.4;osmolality, 325mOsmkg H20f)or 60min. Fig. 1illustrates theeffects of a hypotonic solution (i.e. reduction of the bath osmolality from 325 to 165 mOsmkg H20 by removing sucrose from the isotonic solution) on cell volume. 2.7,KCl; 2.7,EGTA; 5.6,glucose; 1 Na,-ATP; pH 7.4,0.1%bovine serum thatcontributesto RVD, we conducted cell-attached patch albumin, 1 unitfml streptolysin 0,and 1 n~ phalloidin for 5 min at clamp recordingson the apical membrane.Fig. 2 illustrates the. Were rarely observed (
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