to avoid excessive alterations of cell volume that may   jeopardize structural integrity and a variety of cellular   functions , mammalian cells are able to precisely maintain their size in the face of osmotic perturbations   through the regulated loss or gain of intracellular ions   or other osmolytes ( nilius et al . ; okada , 1997 ; lang et al . , 1998 ) . even under isotonic conditions , volume constancy of any mammalian   cell is challenged by the transport of osmotically active   substances across the cell membrane and alterations in   cellular osmolarity by metabolism ( lang et al . , 1998 ) . thus , the continued operation of cell volume regulatory mechanisms , such as volume - regulated chloride   ( cl ) currents ( icl.vol ) , is required for cell volume homeostasis in many mammalian cells ( nilius et al . , 1996 ;   strange et al . , 1996 ; okada , 1997 ) . we have recently   provided evidence that the channel protein responsible for icl.vol in the heart and many other mammalian   cells is encoded by the clc-3 gene ( duan et al . clc-3 belongs to the large gene   family of clc cl channels that are comprised of 12 putative transmembrane - spanning domains ( kawasaki et al . ,   1994 ; kawasaki et al . , 1995 ; jentsch , 1996 ; schmidt - rose and jentsch , 1997 ) . expressed clc-3 cl channels   in oocytes and mammalian cells , 1994 , 1995 ; duan   et al . , 1997b ) and hypertonic cell shrinkage while they   are activated by hypotonic cell swelling ( duan et al . little is currently known , however , about the   molecular mechanisms of regulation of icl.vol by cell volume ( okada , 1997 ; strange , 1998 ; clapham , 1998 ) . alternations of cell volume during extra- and intracellular osmotic perturbation trigger a multitude of intracellular signaling events , including various second   message cascades , phosphorylation or dephosphorylation of target proteins , as well as altered gene expression ( waldegger et al . cell   swelling has been shown to induce protein dephosphorylation , which in turn activates k - cl cotransport ( jennings and al - rohil , 1990 ; jennings and schulz , 1991 ;   bize and dunham , 1994 ; starke and jennings , 1993 )   and inhibits na - k-2cl cotransport ( klein et al . , 1993 ;   haas et al . , 1995 ; lytle , 1998 ) . this swelling - induced   protein dephosphorylation may be due to decreased kinase activity ( jennings and al - rohil , 1990 ; bize and   dunham , 1994 ; gibson and hall , 1995 ) and/or increased activities of serine / threonine protein phosphatases ( pps , probably pp1 and pp2a ) ( jennings and   schulz , 1991 ; starke and jennings , 1993 ; lytle , 1998 ) . on the other hand , cell shrinkage has been shown to   cause protein phosphorylation , which in turn inhibits   k - cl cotransport ( jennings and al - rohil , 1990 ; jennings   and schulz , 1991 ; bize and dunham , 1994 ; starke and   jennings , 1993 ) and activates na - k-2cl cotransport   ( klein et al . , 1993 ; in   ehrlich mouse ascites tumour cells , cell shrinkage   causes a rapid increase ( 174% within 1 min ) in pkc activity in the membrane fraction that appears to be involved in the activation of the na - k-2cl cotransport after cell shrinkage ( larsen et al . , 1994 ) . pkc - dependent   phosphorylation is also involved in the cell shrinkage   activation of a nonselective conductance in caco-2 cells   ( nelson et al . , in fact , cell swelling and shrinkage have been shown to induce protein dephosphorylation and phosphorylation , respectively , in a variety of   cell systems ( grinstein et al . , 1992 ; palfrey , 1994 ) , including epithelial ( haas et al . , 1995 ) and endothelial   ( santell et al . , 1993 ; klein et al . , 1993 ) cells , erythrocytes ( jennings and al - rohil , 1990 ; jennings and schulz ,   1991 ; starke and jennings , 1993 ; lytle , 1998 ) , ehrlich   mouse ascites tumour cells ( larsen et al . , 1994 ; krarup   et al . , 1998 ) , and cardiac myocytes ( hall et al . , 1995 ) . therefore , these studies all suggest that phosphorylation / dephosphorylation of proteins ( such as ionic   channels and transportors ) due to altered protein kinase and/or phosphatase activities may be a common   process linking changes in cell volume to protein functions . it is noteworthy that native icl.vol in heart and many   other tissues is also strongly regulated by phosphorylation / dephosphorylation . activation of intracellular   pkc ( duan et al . , 1995 ; hardy et al . , 1995 ; coca - prados   et al . , 1996 ; bond et al . , 1998 ; , 1998 ) and inhibition of pps ( hall et al . , 1995 ; doroshenko , 1998 ) both strongly inhibit icl.vol even under hypotonic conditions , while inhibition of pkc can activate icl.vol under   isotonic conditions ( coca - prados et al . , 1995 , 1996 ; dick et al . , 1998 ) . to study the volume - sensing mechanism of clc-3 channels and its potential linkage to   channel phosphorylation , we used a variety of approaches from the tight - seal whole - cell voltage - clamp   study of pharmacological agents on native and cloned   channels to analysis of the functional expression of   wild - type and site - directed mutants of clc-3 channels   in nih/3t3 cells . all of the results in this study are   consistent with the identification of the volume sensor   as a distinct serine residue in a consensus pkc - phosphorylation site in the intracellular amino terminus of   clc-3 chloride channel . these results thus provide a   new structural link between protein function ( channel   activity ) and alterations in phosphorylation  dephosphorylation in response to changes in cell volume during osmotic perturbations . the serine at position 51 and/or 362 was altered by a s51 , s362 ,   and s51 + s362 to an alanine site - specific mutation introduced   into gpclc-3 cdna ( deng and nickoloff , 1992 ) . the mutation   was confirmed by nucleotide sequencing of both strands of the   mutated cdna . nih/3t3 cells were transiently transfected by   electroporation as previously described ( duan et al . , 1997b ) . each dish was transfected with appropriate combinations of cd8   ( a lymphocyte cell surface antigen ) in the h3-cd8 plasmid construct as a marker for transfection ( 4 g ) and wtclc-3 , s51aclc-3 ,   s362aclc-3 , or s51a transfected cells were identified by their binding to cd8-coated beads ( m-450 cd8 ; dyna - beads ) . currents were measured from isolated nih/3t3 cells or guinea - pig atrial and ventricular myocytes at room temperature ( 22 24c ) by the tight - seal whole - cell voltage - clamp technique as described ( hamill et al . , 1981 ; duan et al . , 1997a , b ) . to obtain   whole - cell current  voltage relations , cells were held at 40 mv   and test potentials were applied from 100 to + 120 mv for 400   ms in + 20-mv increments at an interval of 5 s ( voltage - clamp   protocol is shown in fig . current amplitudes were measured at 8 ms after the corresponding voltage step relative to 0   current level and normalized to cell capacitance ( pa pf ) . to   obtain time - dependent changes in current amplitude before and   after different interventions , cells were clamped from a holding   potential of 40 mv to hyperpolarizing potential of 100 mv   for 100 ms , back to 40 mv for 10 ms , and then to a depolarizing   potential of + 100 mv for 100 ms ( voltage - clamp protocol is   shown in fig . the same hyperpolarizing and depolarizing pulses were imposed every 30 s. all results are expressed as   mean  sem . statistical comparisons were performed by student 's t test and a two - tailed probability of < 5% was taken to indicate statistical significance . the hypotonic ( 250 mosm / kg h2o , measured by   freezing point depression , osmomette ; precision systems inc . )   bath solutions for recording in nih/3t3 cells contained ( mm ) :   125 nacl , 2.5 mgcl2 , 2.5 cacl2 , 10 hepes , ph 7.2 , [ cl]o = 135   mm . the isotonic and hypertonic bath solutions were the same   as the hypotonic solution except that the osmolarity was adjusted   to 300 and 350 mosm / kg h2o , respectively , with mannitol .   when experiments were performed with decreased [ cl]o , iodide   ( i ) or aspartate ( asp ) was used to replace cl at equimolar   concentration ( 110 mm ) . the pipette ( internal ) solution for recordings in nih/3t3 contained ( mm ) : 135 n - methyl - d - glucamine chloride ( nmdg - cl ) , 2 egta , 5 mg - atp , 10 hepes , ph   7.2 , [ cl]i = 135 mm , 300 mosm / kg h2o using mannitol ) . the   hypotonic ( 220 mosm / kg h2o ) bath solutions for recording in   cardiac myocytes contained ( mm ) : 90 nacl , 0.8 mgcl2 , 1.0 cacl2 ,   0.2 cdcl2 , 2.0 bacl2 , 0.33 nah2po4 , 10 tetraethylammonium - cl ,   10 hepes , 5.5 glucose , ph 7.4 , [ cl]o = 108 mm . the isotonic   bath solutions were the same as the hypotonic solution except   that the osmolarity was adjusted to 300 mosm / kg h2o , respectively , with mannitol . when low [ cl]o was needed , nai or na - aspartate was used to replace nacl at equimolar concentration   ( 90 mm ) . the pipette ( internal ) solution for recordings in cardiac myocytes contained ( mm ) : 108 nmdg - cl , 2 egta , 5 mg - atp , 10 hepes , ph 7.4 , [ cl]i = 108 mm , 290 mosm / kg h2o using mannitol . in some experiments , cell diameters were measured continuously using a video edge detector ( crescent electronics ) and cell volumes were calculated assuming a simple   spherical geometry . all chemicals were from sigma chemical co.   phorbol 12,13-dibutyrate ( pdbu ) , bisindolylmaleimide i - hcl   ( bim ) , okadaic acid , and calyculin a were obtained from calbiochem corp . and prepared as stock solutions of 1 or 10 mm in   dimethyl sulfoxide and added to known volume of superfusion   solutions to produce the desired concentrations . the serine at position 51 and/or 362 was altered by a s51 , s362 ,   and s51 + s362 to an alanine site - specific mutation introduced   into gpclc-3 cdna ( deng and nickoloff , 1992 ) . the mutation   was confirmed by nucleotide sequencing of both strands of the   mutated cdna . nih/3t3 cells were transiently transfected by   electroporation as previously described ( duan et al . , 1997b ) . each dish was transfected with appropriate combinations of cd8   ( a lymphocyte cell surface antigen ) in the h3-cd8 plasmid construct as a marker for transfection ( 4 g ) and wtclc-3 , s51aclc-3 ,   s362aclc-3 , or s51a transfected cells were identified by their binding to cd8-coated beads ( m-450 cd8 ; dyna - beads ) . currents were measured from isolated nih/3t3 cells or guinea - pig atrial and ventricular myocytes at room temperature ( 22 24c ) by the tight - seal whole - cell voltage - clamp technique as described ( hamill et al . , 1981 ; duan et al . , 1997a , b ) . to obtain   whole - cell current  voltage relations , cells were held at 40 mv   and test potentials were applied from 100 to + 120 mv for 400   ms in + 20-mv increments at an interval of 5 s ( voltage - clamp   protocol is shown in fig . current amplitudes were measured at 8 ms after the corresponding voltage step relative to 0   current level and normalized to cell capacitance ( pa pf ) . to   obtain time - dependent changes in current amplitude before and   after different interventions , cells were clamped from a holding   potential of 40 mv to hyperpolarizing potential of 100 mv   for 100 ms , back to 40 mv for 10 ms , and then to a depolarizing   potential of + 100 mv for 100 ms ( voltage - clamp protocol is   shown in fig . the same hyperpolarizing and depolarizing pulses were imposed every 30 s. all results are expressed as   mean  sem . statistical comparisons were performed by student 's t test and a two - tailed probability of < 5% was taken to indicate statistical significance . the hypotonic ( 250 mosm / kg h2o , measured by   freezing point depression , osmomette ; precision systems inc . )   bath solutions for recording in nih/3t3 cells contained ( mm ) :   125 nacl , 2.5 mgcl2 , 2.5 cacl2 , 10 hepes , ph 7.2 , [ cl]o = 135   mm . the isotonic and hypertonic bath solutions were the same   as the hypotonic solution except that the osmolarity was adjusted   to 300 and 350 mosm / kg h2o , respectively , with mannitol . when experiments were performed with decreased [ cl]o , iodide   ( i ) or aspartate ( asp ) was used to replace cl at equimolar   concentration ( 110 mm ) . the pipette ( internal ) solution for recordings in nih/3t3 contained ( mm ) : 135 n - methyl - d - glucamine chloride ( nmdg - cl ) , 2 egta , 5 mg - atp , 10 hepes , ph   7.2 , [ cl]i = 135 mm , 300 mosm / kg h2o using mannitol ) . the   hypotonic ( 220 mosm / kg h2o ) bath solutions for recording in   cardiac myocytes contained ( mm ) : 90 nacl , 0.8 mgcl2 , 1.0 cacl2 ,   0.2 cdcl2 , 2.0 bacl2 , 0.33 nah2po4 , 10 tetraethylammonium - cl ,   10 hepes , 5.5 glucose , ph 7.4 , [ cl]o = 108 mm . the isotonic   bath solutions were the same as the hypotonic solution except   that the osmolarity was adjusted to 300 mosm / kg h2o , respectively , with mannitol . when low [ cl]o was needed , nai or na - aspartate was used to replace nacl at equimolar concentration   ( 90 mm ) . the pipette ( internal ) solution for recordings in cardiac myocytes contained ( mm ) : 108 nmdg - cl , 2 egta , 5 mg - atp , 10 hepes , ph 7.4 , [ cl]i = 108 mm , 290 mosm / kg h2o using mannitol . in some experiments , cell diameters were measured continuously using a video edge detector ( crescent electronics ) and cell volumes were calculated assuming a simple   spherical geometry . all chemicals were from sigma chemical co.   phorbol 12,13-dibutyrate ( pdbu ) , bisindolylmaleimide i - hcl   ( bim ) , okadaic acid , and calyculin a were obtained from calbiochem corp . and prepared as stock solutions of 1 or 10 mm in   dimethyl sulfoxide and added to known volume of superfusion   solutions to produce the desired concentrations . under isotonic ( 300 mosm / kg h2o ) symmetrical cl   ( 135 mm ) conditions , wtclc-3transfected cells generated basally active outwardly rectifying whole - cell currents ( fig . 1 a , a ) with a mean current density of 442    38 pa pf at + 80 mv and 253  27 pa pf at 80   mv and a mean reversal potential of 1.8  0.3 mv ( n =   6 ) . exposure of these cells to hypotonic solutions ( 250   mosm / kg h2o , 17% hypotonic ) for > 2 min caused   significant cell swelling and increased the membrane   current densities to 946  56 pa pf at + 80 mv and   593  46 pa pf at 80 mv ( n = 6 ) due to an increase in the number of active channels ( strange et al . ,   1996 ; duan et al . 1 a , b ) . these results   indicate that under basal isotonic conditions , most expressed clc-3 channels remain in a closed state that   can be activated by hypotonic cell swelling , suggesting   the possible existence of an endogenous cytosolic inhibitor under isotonic conditions ( krick et al . , activation of pkc by pdbu ( 100   nm ) under hypotonic conditions strongly inhibited the   currents ( fig . 1 a , c ) in a voltage - independent fashion as   previously described ( duan et al . , 1997b ) , while hypotonic solutions induced a similar increase in cell volume   in control ( 129  5.4% , n = 8) and pdbu - containing   solutions ( 122  2.0% , n = 7 , p = ns ) . downregulation of endogenous pkc by exposure of wtclc-3transfected nih/3t3 cells to pdbu ( 1 m ) for > 24 h   ( duan et al . , 1995 ; pears and goode , 1997 ) not only   abolished the inhibition of wtclc-3 currents by acute   application of pdbu ( fig . 1 b , c ) , but also , surprisingly ,   changed the volume sensitivity of these channels . in   downregulated cells under isotonic conditions , most   channels were constitutively open with a mean current   density of 1,009  92 pa pf at + 80 mv and 678    60 pa pf at 80 mv ( n = 4 ) ( fig . 1 b , a ) and subsequent hypotonic cell swelling failed to further significantly increase current densities ( fig . 1 b , b ) in these   cells ( 1,039  91 pa pf at + 80 mv and 701  70   pa pf at 80 mv , p = ns ) . inhibition of endogenous pkc in wtclc-3transfected cells by acute application of bim ( 100 nm ) , a highly selective pkc inhibitor   ( toullec et al . , 1991 ) , also dramatically increased membrane cl current densities under isotonic conditions   ( fig . 1 c , a and b ) and abolished further activation of   expressed channels by cell swelling ( fig . 1 c , c ) . the   endogenous cl currents in nih/3t3 cells , while also   volume sensitive , contribute very little to the results described above ( duan et al . these results strongly suggest that endogenous   pkc in these nih/3t3 cells is a strong cytosolic inhibitor of clc-3 channels and that relief of pkc inhibition   may be linked to hypotonic - induced opening of the   channel . to further test this hypothesis , we performed   similar experiments in isolated guinea - pig atrial and   ventricular cells from which the clc-3 gene was originally cloned ( duan et al . 2 ,   both the basally active and swelling - activated currents   in atrial ( fig . identical to the cloned gpclc-3 channel expressed in nih/ 3t3 cells ( duan et al . , 1997b ) , these cell swelling- induced and pkc - sensitive currents in both atrial ( fig .   2 c ) and ventricular ( fig . 2 d ) myocytes had an anion   selectivity of i > cl > > asp . consistent with our observations in nih/3t3 cells , bim - induced inhibition of   endogenous pkc also activated native icl.vol in both   atrial ( fig . 3 a , b ) and ventricular ( fig . 3 b , b ) myocytes under isotonic conditions and prevented further   activation by subsequent hypotonic cell swelling ( fig . 3 ,   a , c and b , c ) . as shown in fig . 3 c , the swelling- and   bim - induced currents were inhibited by extracellular   4,4-diisothiocyanostilbene-2,2-disulfonate ( dids ) ( 100   m , fig . 3 c , a and b ) and atp ( 10 mm , fig . 3 c , c ) in   a characteristic voltage - dependent manner that closely   resembles the inhibition of native icl.vol by these compounds in a wide variety of cells ( duan et al . , 1996 ; okada , 1997 ; yamazaki et al . , 1998 ) and clc-3   currents in oocytes and mammalian cells ( kawasaki et al . these results support the idea   that pkc phosphorylation and dephosphorylation of   both the wtclc-3 and native channel protein play a crucial role in channel regulation by changes in cell volume .    in intact cells , processes that are reversibly controlled   by protein phosphorylation require not only a protein   kinase but also a protein phosphatase ( hunter , 1995 ) . the net level of protein phosphorylation depends on   the balance of kinase and phosphatase activities ( cohen , 1992 ) , and both protein kinases and phosphatases   have been reported to be subject to regulation by cell   volume ( jennings and al - rohil , 1990 ; jennings and   schulz , 1991 ; starke and jennings , 1993 ; waldegger et al . in fact , inhibition of serine / threonine protein phosphatases by calyculin a in nih/3t3   cells causes not only a marked increase in protein phosphorylation in both cytosolic and insoluble cellular   fractions , but also a reversible cell shape change   ( chartier et al . , 1991 ) . to further test the hypothesis   that a balance between channel protein phosphorylation and dephosphorylation may be the key regulatory   event responsible for clc-3 channel regulation by cell   volume in the face of osmotic perturbation , we studied   the effects of two highly potent serine / threonine protein phosphatase inhibitors , okadaic acid ( cohen et al . ,   1990 ) and 1989 ) , on wtclc-3 channels expressed in nih/3t3 cells . as shown   in fig . 4 , both okadaic acid ( 100 nm , a ) and calyculin a   ( 20 nm , b ) not only inhibited basally active wtclc-3   channels under isotonic conditions , but also prevented   hypotonic cell - swelling activation of these channels . similar results were observed in four ( okadaic acid )   and five ( calyculin a ) different cells in which wtclc-3   were stably or transiently transfected . both basally and   swelling - activated clc-3 channels were always strongly   inhibited by either okadaic acid or calyculin a , when   added before or after induction of cell swelling , indicating that pps are continuously involved in channel   regulation and the balance of pkc - pp activity is constantly regulated by cell volume . the apparent link between pkc phosphorylation dephosphorylation and cell swelling  induced activation of clc-3 channels prompted a consideration of putative protein pkc phosphorylation sites as potential   volume sensors ( kawasaki et al . , 1994 ; one is serine 51   near the amino terminus ( arg - arg - lle - asn - ser - lys - lys - lys ) and the other is serine 362 within the cytoplasmic   loop between transmembrane domains d7 and d8   ( arg - arg - lys - ser - thr - lys ) . we tested the hypothesis   that the inhibition , by pkc activators and phosphatase   inhibitors , and the activation , by hypotonic cell swelling and pkc inhibitors , of clc-3 channels may be due   to direct phosphorylation serine 51 and/or serine 362 were changed to alanines and three mutants were generated : single mutants s51a , s362a , and double mutant s51a + s362a   ( fig . 5 a ) . results of whole - cell patch - clamp recording   from these three clc-3 mutants transfected into 3t3   cells are shown in fig . 5 , b  d . cells were exposed   either to isotonic ( a ) , hypotonic ( b ) , or hypotonic   pdbu 100 m ( c ) solutions . the double mutant   s51a+s362aclc-3 generated a large constitutively activate iclc-3 with a mean current density of 918  23 pa   pf at + 80 mv and 557  48 pa pf at 80 mv   ( n = 5 ) ( fig . 5 b , a ) that was not significantly augmented ( mean current density of 970  48 pa pf at   + 80 mv and 629  85 pa pf at 80 mv , p = ns )   by hypotonic cell swelling ( fig . 5 b , b ) or inhibited by   pdbu - induced activation of pkc ( fig . 5 b , c , mean current density of 952  41 pa pf at + 80 mv and 606    71 pa pf at 80 mv , p = ns ) . most channels were open even under isotonic conditions and   generated a large constitutively active iclc-3 ( fig . 5 c , a ,   mean current density of 930  18 pa pf at + 80 mv   and 608  22 pa pf at 80 mv , n = 7 ) that was no   longer responsive to cell swelling ( fig . 5 c , b , mean   current density of 971  27 pa pf at + 80 mv and   650  23 pa pf at 80 mv , p = ns , compared with   isotonic conditions ) or pdbu ( fig . 5 c , d , mean current density of 907  31 pa pf at + 80 mv and 627    31 pa pf at 80 mv , p = ns compared with isotonic   and hypotonic conditions ) . as shown in fig . 5 d , however , the s362aclc-3 mutant yielded a channel with an   intermediate responsiveness to cell swelling and pkc . the mean current densities of s362aclc-3 under isotonic conditions were 610  42 pa pf at + 80 mv and   348  31 pa pf at 80 mv ( n = 6 ) that are significantly higher ( p < 0.05 ) than the mean current densities of wtclc-3 under the same isotonic conditions . exposure of these s362aclc-3transfected cells to hypotonic solutions caused a further increase in current   densities ( 912  90 pa pf at + 80 mv and 468  37   pa pf at 80 mv , n = 6 , p < 0.05 compared with isotonic conditions ) . activation of pkc by pdbu under   hypotonic conditions caused significantly less inhibition of s362aclc-3 current than that of wtclc-3 ( 55    7% inhibition of s362aclc-3 currents [ n = 6 ] vs. 81    3% inhibition of wtclc-3 currents [ n = 6 ] at + 80 mv ,   p < 0.01 ) . we also performed blinded experiments in   which coded nih/3t3 cells transiently transfected with   cd8 alone ( control ) , wtclc-3 , s51aclc-3 , s362aclc-3 ,   and s51a+s362aclc-3 mutants were used in a randomized fashion to study the response of expressed   currents to hypotonic cell swelling and pkc activation . 6 compares the mean current densities of cells   transiently transfected with cd8 alone ( control ) , wtclc-3 , s51aclc-3 , s362aclc-3 , and s51a+s362aclc-3   mutants at + 80 mv under isotonic ( a ) , hypotonic ( b ) ,   and hypotonic pdbu 100 nm ( c ) conditions , respectively . while only very small currents could be detected   from cd8-transfected cells under isotonic and hypotonic conditions with similar densities as reported before ( duan et al . , 1997b ) , it is very clear that wtclc-3   and all three clc-3mutant transfected cells elicited   significantly larger basally active outwardly rectifying   currents under isotonic conditions . wild - type clc-3   channels were activated by cell swelling and inhibited   by pkc activation , as previously shown ( fig . both s51aclc-3 and s51a+s362aclc-3 channels were   constitutively opened under isotonic conditions and   thus generated significantly larger a ) . these phenotypes exhibited no significant response to changes in cell volume ( fig . basal s362aclc-3 currents were larger than wtclc-3 ( p < 0.05 ) but smaller   than s51aclc-3 and s51a+s362aclc-3 ( p < 0.05 )   ( fig . 6 a ) . as shown   in fig . 7 a , a , hypertonic ( 350 mosm / kg h2o ) cell   shrinkage also failed to inhibit s51aclc-3 channels   ( 898  32 pa pf under isotonic conditions vs. 869    48 pa pf under hypertonic conditions , n = 5 , p =   ns ) . a , d ) all blocked s51aclc-3 with a characteristic voltage dependence closely resembling their   effects on wtclc-3 ( duan et al . , 1997b ; yamazaki et al . ,   1998 ) . table i summarizes the shifts   in the reversal potentials of native icl.vol in guinea - pig   atrial and ventricular myocytes and wild - type and mutant clc-3 channels in nih/3t3 cells induced by i or   asp and their relative permeabilities . the permeability ratios were calculated from the shifts using the modified goldman - hodgkin - katz equation ( hille , 1992 )   for monovalent anion substitutions . all three mutants   of clc-3 had a similar permeability ratio to i or asp   with respect to cl and an identical anion - selectivity of   i > cl it has been reported that rat clc-3 channels may be   inhibited by increases in intracellular ca when expressed in chinese hamster ovary cells ( kawasaki et al . ,   1995 ) . similarly , exposure of guinea - pig cardiac wtclc-3 transfected cells to the ca ionophore , ionomycin ( 1   m ) , also caused a dramatic inhibition of wtclc-3 currents under hypotonic conditions ( fig . 6 b , however , the inhibition of gpclc-3   currents by ionomycin was prevented by inhibition of   endogenous pkc when cells were pretreated with bim   ( 100 nm ) . these results suggest that inhibition of gpclc-3 channels by an increase in intracellular ca may   be due to a ca - sensitive pkc - phosphorylation  mediated mechanism . in which the ca inhibition of clc-3 channels in inside - out membrane patches   from chinese hamster ovary cells was reported to be   phosphorylation independent ( kawasaki et al . , 1995 ) . both ca - sensitive ( pkc ) and -insensitive ( pkc and pkc )   pkc isozymes are abundantly expressed in nih/3t3   cells ( szallasi et al . , 1994 ) , and bim inhibits all pkc ,   i , ii ,  ,  , and  isozymes ( gekeler et al . , 1996 ; toullec et al . , 1991 ) . thus , it is possible that both ca - sensitive and -insensitive pkc isozymes can phosphorylate   the clc-3 protein and cause similar conformational   changes to close the channel . to further examine   whether the inhibition of gpclc-3 channels by intracellular ca is dependent on pkc phosphorylation of the   channel , we performed further experiments to study   the response of expressed mutant s51aclc-3 channel to   ionomycin . while the properties of s51aclc-3 channels under isotonic and hypotonic conditions ( fig . 8 c ,   a and b ) were identical to those shown in fig . 5 c , a and   b , respectively , ionomycin failed to inhibit s51aclc-3   channels ( fig . 8 c , c and d ) , confirming the involvement of amino - terminal pkc - phosphorylation in the   ca inhibition of clc-3 channels . in this study , we examined the molecular mechanism   responsible for the activation of volume - regulated cl   channels by hypotonic cell swelling using both cloned   guinea - pig cardiac clc-3 expressed in nih/3t3 cells   and native icl.vol currents in guinea - pig atrial and ventricular myocytes . our functional and mutational studies of the clc-3 gene product indicate that the activation of clc-3 channels during hypotonic - induced cell   swelling is attributable to relief of endogenous pkc inhibition of these channels caused by cell swelling induced dephosphorylation of a serine residue within   the amino terminus of the channel protein . thus , these   results provide an important new clue into the molecular link between changes in cell volume , protein phosphorylation protein phosphorylation or dephosphorylation is a   common rapid and reversible means of transducing signals from the extracellular environment to many cellular responses ( witters , 1990 ; hunter , 1995 ) . it should   not be surprising that cells are able to use these rapid   and reversible means to control their sizes . how   changes in cell volume are linked to changes in activity   of protein kinases or phosphatases is still not clear . one   of the most acute signaling events triggered by osmotic   challenge may be dilution or concentration of cellular   constituents including proteins leading to changes in   intracellular macromolecular crowding and confinement , which may profoundly alter kinase - phosphatase   activities ( fulton , 1982 ; jennings and al - rohil , 1990 ;   jennings and schulz , 1991 ; minton et al . , 1992 ; starke   and jennings , 1993 ; garner and burg , 1994 ) . alternatively , slower subacute signaling events caused by cell   swelling or shrinkage may be related to changes in the   synthesis of second messengers . for example , exposure   of mammalian and plant cells to acute hyperosmotic   stress stimulates rapid synthesis of phosphatidylinositol-3,5-bisphosphate , a new phosphoinositide second messenger in the phospholipase c  pkc cascade that may   associate with the cytoskeleton ( dove et al . , 1994 ,   1997 ) . finally , the slowest and long - term intracellular   signaling events involved in continuous volume regulation may be due to alterations in gene expression of   second messengers ( waldegger et al . the exact interaction between   cell volume and elements of intracellular signaling and   detailed intermediate processes of how pkc and pp activities may be regulated by cell volume requires further study . our data suggests that clc-3 channels may exist in either a closed phosphorylated state or an active dephosphorylated state . pkc phosphorylation of the nh2 terminus of clc-3 channel may cause a crucial change in   channel conformation and close the channel pore . combined with data from our previous studies and   those of other laboratories ( li et al . , 1989 ; jennings   and al - rohil , 1990 ; witters , 1990 ; garner and burg ,   1994 ; duan et al . , 1995 , 1997a , b ; coca - prados et al . ,   1995 , 1996 ; strange et al . , 1996 ; dove et al . , 1997 ;   waldegger et al . , 1997a ) , we propose that clc-3 channels are continuously controlled by a volume - sensitive phosphorylation  dephosphorylation reaction mediated by pkc ( both ca - sensitive and -insensitive   isozymes ) and pps ( probably pp1 and pp2a ) . under   isotonic conditions , a balance of basal pkc and pp activities usually keeps most clc-3 channels in a phosphorylated closed state and only few channels are in   the dephosphorylated open state . these few active   channels generate a  basal  current ( duan et al . , 1992 ,   1995 , 1997a , b ; liu et al . , 1993 ; coca - prados et al . , 1995 ;   voets et al . , 1996 ; dick et al . , 1998 ) . under hypotonic   conditions , pkc activity is diminished due possibly to   dilution ( jennings and al - rohil , 1990 ; garner and   burg , 1994 ) , redistribution or alteration in pkc or pp   activity ( jennings and schulz , 1991 ; starke and jennings , 1993 ; palfrey , 1994 ; lytle , 1998 ) ; clc-3 channels   become dephosphorylated and more channels open ,   producing a larger macroscopic current ( duan et al . ,   1995 , 1997a , b ; strange et al . , 1996 ) . under hypertonic   conditions , pkc activity may be increased ( jennings   and al - rohil , 1990 ; larsen et al . , 1994 ; , 1998 ; dove et al . , 1997 ;   waldegger et al . , 1997a , b ) and pp activity may be diminished ( palfrey , 1994 ; lytle , 1998 ) , thus more channels become phosphorylated and close . therefore , it is   proposed that serine 51 , a putative pkc phosphorylation site near the nh2 terminus of clc-3 , may represent   an important volume sensor of the channel that directly links channel activity to alterations in intracellular   pkc - pp activity . pkc activation has been previously reported to inhibit voltage - dependent cl conductances in hippocampal pyramidal cells ( madison et al . , 1986 ) , resting   cl conductance in vascular smooth muscle cells ( saigusa and kokubun , 1988 ) , skeletal muscle cells ( brinkmeier and jockusch , 1987 ) , clc-1 in skeletal muscle   and hek cell expression system ( rosenbohm et al . ,   1995 ) , and the hyperpolarization - activated clc-2 cl   channel that is also regulated by cell volume ( staley ,   1994 ; staley et al . , 1996 ; fritsch and edelman , 1996 ,   1997 ) . consistent with our finding in native guinea - pig   atrial and ventricular myocytes and cloned clc-3transfected nih/3t3 cells , pkc activation inhibits icl.vol and   outwardly rectifying chloride channel in rabbit atrial   myocytes ( duan et al . , 1995 ) , 1995 ) , human airway epithelial cells   ( li et al . , 1989 ) , and canine visceral smooth muscle   cells ( dick et al . , 1998 ) . inhibition of pps has also been   shown to inhibit icl.vol in chick heart cells ( hall et al . recent studies in human nonpigmented ciliary   epithelial cells and canine colonic smooth muscle cells   have also shown that pkc inhibitors isosmotically upregulated icl.vol ( coca - prados et al . , 1995 , 1996 ; dick et   al . , dephosphorylation and cell swelling also activate a voltage - gated cl channel in ascidian embryos   ( villaz et al . , 1995 ) . inhibition of pkc also activates the   volume - sensitive clc-2 channel in human intestinal   t84 epithelial cells ( fritsch and edelman , 1996 ) . our   results provide direct molecular and pharmacological   evidence indicating that channel phosphorylation dephosphorylation plays a crucial role in regulation of   volume sensitivity of clc-3 channels and native icl.vol . therefore , these data provide further evidence that   protein kinases and phosphatases may be secondary   mediators of a subset of cellular responses to cell volume changes that directly control the function of proteins such as clc-3 channels . it has also been reported , however , that icl.vol in some   tissues is either stimulated by phorbol esters , presumably through activation of pkc , or not regulated by   pkc activators and inhibitors ( jackson and strange ,   1993 ; szcs et al . , 1996 ; miwa et al . , 1997 ; reviewed by   strange et al . , 1996 ; okada , 1997 ; and strange , 1998 ) .   while most of these studies did not directly measure   pkc activity , miwa et al . ( 1997 ) reported that phorbol   ester tpa ( 12-o - tetradecanoylphorbol-13-acetate ) still   had no effect on icl.vol in human epidermoid kb cells   even though activation of pkc by this compound could   be biochemically proven . it is possible , however , that the intrinsic response of cells to phorbol esters in terms of activation of different pkc isozymes may vary from cell to   cell ( nishizuka , 1988 ; hug and sarre , 1993 ) and the   phosphorylation of clc-3 chloride channel may be mediated by specific pkc isozymes . another possibility is   that the so called  pkc activators  ( phorbol esters ) and    pkc inhibitors  may also act on unknown protein kinases other than pkc . it may be possible that the kinase involved in regulation of clc-3 is not simply pkc   but another serine / threonine kinase acting at the same   s51 . ( 1997a ) have recently cloned a   volume - regulated serine / threonine protein kinase designated h - sgk , which is upregulated by hypertonic cell   shrinkage and depressed by hypotonic cell swelling . h - sgk has 50% homology throughout its catalytic domain with pkc and is widely expressed in many tissues .   however , gene transcription and translation of h - sgk   may be too slow to account for the change in icl.vol . simple biochemical experiments or attempts to measure   activities or translocations of pkc or pkc isozymes may   not be sufficient to provide a definitive answer to the   complex question whether or not pkc is in fact activated by cell shrinkage and inhibited by swelling . obviously , more extensive experiments will be needed to   detect rapid but more subtle changes in pkc or other   protein kinases or phosphatases near the membrane   during volume alterations . although our mutation experiments on the consensus pkc phosphorylation sites   provide strong evidence supporting the role of pkc in   the volume regulation of the channel , direct measurement of changes in kinase and/or phosphatase activity   and biochemical evidence for phosphorylation of clc-3   during alternations of cell volume are needed . it   should also be pointed out that consensus phosphorylation sites of a protein can be promiscuous and mutations can alter the conformation of the protein independent of phosphorylation . in some cells , the mechanism of channel activation by cell swelling may be more   complicated than described here since it is also possible that the volume - regulated cl channels in some native cells may actually be composed of heterodimers ;   e.g. , clc-3 along with other clc subunits ( lorenz et al . ,   1996 ) . whether a non  pkc - regulated but volume - sensitive clc-3 isoform , or heteromultimer , or other members of the clc-3/clc-4/clc-5 subbranch ( jentsch ,   1996 ) are also involved in cell - volume regulation and   account for icl.vol in other cell types needs further investigation . relations between   pkc activity and volume regulation of wtclc-3 in nih/3t3   cells . ( a ) pkc activation inhibited both basally active and hypotonic cell - swelling  induced iclc-3 .   when overexpressed in nih/ 3t3 cells , only a small portion of   the clc-3 channels were active   under isotonic conditions ( a ) . subsequent exposure of the   same cell to hypotonic solutions   caused a further increase in current amplitude ( b ) . activation of   pkc by pdbu ( 100 nm ) under   hypotonic conditions caused a   closure of most channels ( c ) . mean current - voltage ( i  v ) curves   from six cells under isotonic (  ) ,   hypotonic (  ) , and hypotonic   pdbu 100 nm (  ) conditions   are shown in d. ( b ) downregulation of endogenous pkc by   exposure of the wtclc-3-transfected nih/3t3 cells to pdbu   1 m for > 24 h not only abolished the inhibition of iclc-3 by   acute application of pdbu ( 100   nm ) , but also changed the sensitivity to changes in osmolality . ( a ) representative current traces   recorded from the pdbu - pretreated clc-3 stably transfected   cells under isotonic conditions . ( b )   subsequent hypotonic cell swelling failed to further increase the   current amplitude of these cells . ( c ) acute application of pdbu   ( 100 nm ) under hypotonic conditions no longer inhibited the   currents . ( d ) mean i  v curves   from four different cells under   isotonic (  ) , hypotonic (  ) , and   hypotonic pdbu 100 nm (  )   conditions . ( c ) inhibition of endogenous pkc by bim ( 100 nm ) activated clc-3 channels in isotonic solutions , and subsequent hypotonic   cell swelling failed to further increase the current density . ( b ) exposure of the same cell to bim ( 100   nm ) under isotonic condition increased the current . ( c ) subsequent exposure of the cell to hypotonic solution caused no further increase   in current amplitude . ( d ) mean i  v curves from five different cells under isotonic (  ) , isotonic bim 100 nm (  ) , and hypotonic bim 100   nm (  ) conditions . inhibition of endogenous pkc by bim ( 100 nm ) activated large outwardly rectifying   cl currents under isotonic conditions in guinea - pig atrial ( a )   and ventricular ( b ) myocytes . subsequent hypotonic cell swelling failed to significantly increase   the current densities in these   cells . ( b ) exposure of the same   cell to bim ( 100 nm ) under isotonic conditions caused a significant increase in the membrane   current with similar biophysical   properties as icl.vol ( see a ) . ( c )   subsequent exposure of the cell   to hypotonic solution caused no   significant increase in current   amplitude . ( d ) mean i  v curves   from six ( c and d ) different cells   under isotonic (  ) , isotonic bim   100 nm (  ) , and hypotonic bim   100 nm (  ) conditions . a and b compare the effects of dids ( 100   m ) on the swelling - induced   icl.vol ( a ) and the bim - induced   icl.vol ( b ) in atrial myocytes . extracellular atp ( 10 mm )   also inhibited bim - induced icl.vol   in a characteristic voltage - dependent manner ( c ) identical to its   inhibitory effect on the wild - type   gpclc-3 channels ( duan et al . ,   1997b ) and icl.vol in many other   tissues and species ( strange et al . ,   1996 ) . comparison of current densities at + 80 mv in nih/ 3t3 cells transfected with cd8 ,   wtclc-3 , s51aclc-3 , s362aclc-3 ,   and s51a+ s362aclc-3 under   isotonic ( a ) , hypotonic ( b ) , and   hypotonic pdbu 100 nm ( c )   conditions , respectively . whole - cell currents were recorded from   cells transfected with different   cdnas that were randomly coded   during experiments . pkc activity and volume - regulation of native icl.vol in   guinea - pig atrial ( a ) and ventricular ( b ) myocytes . ( b ) subsequent exposure   of the same cell to hypotonic solutions caused a further increase   in the currents ( icl.swell ) . ( c ) activation of pkc by pdbu ( 100 nm )   under hypotonic conditions   caused closure of most channels . ( d ) mean i  v relationships from   different atrial ( n = 6 , in a ) or   ventricular ( n = 5 , in b ) cells under isotonic (  ) , hypotonic (  ) ,   and hypotonic pdbu 100 nm   (  ) conditions . ( c and d ) i  v   relationship of whole - cell currents in atrial ( c ) and ventricular ( d ) myocytes before and   after [ cl]o was replaced by   equimolar ( 90 mm ) i or asp   ( ecl = + 46 mv ) . i and asp   substitution of [ cl]o shifted the   reversal potential of icl.vol in atrial   myocytes ( c ) from 2.0  0.5 to   15.3  1.3 and + 39.5  1.7   mv ( n = 6 ) , respectively , and   of icl.vol in ventricular myocytes   ( d ) from 3.2  0.9 to 13.8    2.4 and + 41.2  3.9 mv ( n =   5 ) , respectively . effects of serine / threonine protein phosphatase inhibitors , okadaic acid ( a ) and calyculin a ( b ) , on wtclc-3 channels . whole - cell currents were monitored continuously ( voltage - clamp protocol is shown on the top and described in materials   and methods ) when cells transfected with wtclc-3 were consecutively exposed to isotonic solutions for 10 min , hypotonic solutions   until the changes in current amplitudes reached a steady state   ( 20 min ) , isotonic solutions until the changes in current amplitudes reached a steady state ( 20 min ) , isotonic solutions with 100   nm okadaic acid ( a , oa ) or calyculin a ( b , cla ) for 10 min , hypotonic solutions with oa or cla for 10 min , washout of oa or   cla with hypotonic solutions until currents reached a steady state ,   reapplication of oa or cla in hypotonic solutions and then washout of oa or cla . both oa ( a ) and cla ( b ) not only inhibited   the basally active wtclc-3 channels , but also prevented hypotonic   cell - swelling activation of these channels . both basally and swelling   activated clc-3 channels were always strongly inhibited by both   oa and cla , when added either before or after cell swelling . similar results were observed in four ( oa ) and five ( cla ) different   cells in which wtclc-3 were stably or transiently transfected . effects of pkc on   mutant s51a , s362a , and s51a +   s362a gpclc-3 transiently expressed in nih/3t3 cells . ( a )   transmembrane topology model   for clc cl channels ( schmidt - rose and jentsch , 1997 ) and the   location of mutated residues . ( b d ) whole - cell currents recorded   from three mutants of clc-3 under isotonic ( a ) , hypotonic ( b ) ,   and hypotonic pdbu 100 nm ( c )   conditions , respectively . d show   corresponding mean i  v curves   from different cells transfected   with s51a+s362aclc-3 ( b , d , n   = 5 ) , s51aclc-3 ( c , d , n = 7 ) ,   and s362aclc-3 ( d , d , n = 6 ) ,   respectively . pharmacology and   ion selectivity of clc-3 mutants in   nih/3t3 cells . ( a ) representative whole - cell recording of s51a   clc-3 currents under isotonic   conditions and in the presence   of extracellular hypertonicity ( a ) ,   atp ( b ) , dids ( c ) , and tamoxifen ( d ) . while hypertonic cell   shrinkage failed to inhibit s51a   clc-3 current , extracellular atp   ( 10 mm ) , dids ( 100 m ) , and   tamoxifen ( tmx , 10 m ) all inhibited s51a clc-3 currents in a   characteristic voltage - dependent   manner identical to their inhibitory effects on the wtclc-3 channels ( duan et al . , 1997b ) . v relationship of whole - cell   currents in nih/3t3 cells transfected with s51a + s362a clc-3   ( a ) , s51a clc-3 ( b ) , s362a clc-3   ( c under isotonic conditions and   d under hypotonic conditions )   before and after [ cl]o was replaced by equimolar ( 110 mm )   i or asp ( ecl = + 42.8 mv ) . i   and asp substitution of [ cl]o   shifted the reversal potential of   s51a + s362a clc-3 current ( a )   from 1.0  1.2 to 14.2  1.1   and + 43.8  3.2 mv ( ( b ) from 3.8  0.3 to 14.9    2.6 and + 37.7  1.6 mv ( n = 3 ) ,   respectively , of s362a clc-3 current under isotonic condition ( c )   from 2.3  0.8 to 17.0  2.3   and + 41.0  2.6 mv ( n = 4 ) , respectively , and s362a clc-3 current under hypotonic condition   ( d ) from 2.2  1.1 to 14.5    2.1 and + 38.1  1.5 mv ( n = 3 ) ,   respectively . voltage - clamp protocol is the same as shown on the   top of fig . shifts in reversal potential induced by different anions and their relative permeabilities   values are means  sem ; vr , reversal potential ; px / pcl , permeability ratio   of ion x with respect to cl ; n , number of cells . effects of increase in   intracellular ca on wild - type   and mutant s51aclc-3 currents   in nih/3t3 cells . ( a ) ionomycin ( 1 m ) inhibited wtclc-3   currents under hypotonic conditions in the presence of 2.5 mm   external ca . representative   whole - cell currents recorded under isotonic , hypotonic , and hypotonic ionomycin 1 m conditions and the mean i  v curves of   each ( n = 4 ) are shown in a  d ,   respectively . ( b ) pretreatment of   wtclc-3 transfected nih/3t3   cell with bim ( 100 nm ) under   isotonic condition ( a ) abolished   the upregulation effect by hypotonic cell swelling ( b ) and inhibitory effect by ionomycin ( c ) . ( c ) mutation at   the amino - terminal pkc - phosphorylation site of clc-3   ( s51aclc-3 ) also abolished the   upregulation effect by hypotonic cell swelling ( b ) and inhibitory effect by ionomycin ( c ) . similar results were obtained   from five different cells and the   average i  v curves under each   condition are shown in d.
in many mammalian cells , clc-3 volume - regulated chloride channels maintain a variety of normal   cellular functions during osmotic perturbation . 
 the molecular mechanisms of channel regulation by cell volume ,   however , are unknown . since a number of recent studies point to the involvement of protein phosphorylation / dephosphorylation in the control of volume - regulated ionic transport systems , we studied the relationship between   channel phosphorylation and volume regulation of clc-3 channels using site - directed mutagenesis and patch - clamp techniques . in native cardiac cells and when overexpressed in nih/3t3 cells , clc-3 channels were opened   by cell swelling or inhibition of endogenous pkc , but closed by pkc activation , phosphatase inhibition , or elevation of intracellular ca2 + . 
 site - specific mutational studies indicate that a serine residue ( serine51 ) within a consensus pkc - phosphorylation site in the intracellular amino terminus of the clc-3 channel protein represents an important volume sensor of the channel . 
 these results provide direct molecular and pharmacological evidence indicating that channel phosphorylation / dephosphorylation plays a crucial role in the regulation of volume sensitivity   of recombinant clc-3 channels and their native counterpart , icl.vol .