Characterization of Ca2 -activated Cl- currents in mouse kidney inner medullary - 《American Journal of Physiology》美国生理学杂志干细胞之家



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发表于 2009-4-21 13:40 |只看该作者 |倒序浏览 |打印

作者：Zhiqiang Qu, Raymond W. Wei,  H. Criss Hartzell作者单位：Department of Cell Biology, Emory University School of Medicine, Atlanta,Georgia 30322-3030





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      【摘要】
   Ca 2 -activated Cl - (ClCa) channelswere characterized biophysically and pharmacologically in a mouse kidney inner medullary collecting duct cell line, IMCD-K2. Whole cell recording wasperformed with symmetrical N -methyl- D -glucamine chloride (NMDG)-Cl in the intracellular and extracellular solutions, and theintracellular Ca 2 concentration([Ca 2 ] i ) was adjusted withCa 2 -EGTA buffers. The amplitude of the current wasdependent on [Ca 2 ] i.[Ca 2 ] i rectifying Cl - currents, whereas highCa 2 (21 µM) elicited time-independent currents thatdid not rectify. The currents activated at low [Ca 2 ]exhibited time-dependent activation and deactivation. The affinity of thechannel for Ca 2 was voltage dependent. TheEC 50 for Ca 2 was 0.4 µM at  100 mVand 1.0 µM at -100 mV. The Cl - channel blockerniflumic acid in the bath equally inhibited both inward and outward currentsreversibly, with a K i = 7.6 µM. DIDS,diphenylamine-2-carboxylic acid, and anthracene-9-carboxylic acid reversiblyinhibited outward currents in a voltage-dependent manner. DTT slowly inhibitedthe currents, but tamoxifen did not. Comparing the biophysical andpharmacological properties, we conclude that IMCD-K2 cells express the sametype of ClCa channels as those we have described in detail in Xenopuslaevis oocytes (Qu Z and Hartzell HC. J Biol Chem 276:18423-18429, 2001).
      【关键词】 patch clamp calcium chloride channel biophysics pharmacology5 }" L1 W( v7 V$ N
   C a 2  - ACTIVATED C l - ( C l C a ) channels perform important physiologicalfunctions, including epithelial secretion, repolarization of cardiac actionpotential, regulation of vascular tone, olfactory transduction, neuronalexcitability, and fast block to polyspermy ( 2, 9, 13, 23, 26, 30, 33 ).

The kidney plays a critical role in salt secretion and absorption, andCl - channels are an important component of this process. Determining the roles of different types of Cl - channels isan area of active investigation. The inner medullary collecting duct (IMCD) isthe final segment of the kidney involved in determining acid-base balance andurinary salt composition. In the IMCD, both electrogenic Na absorbtion and electrogenic Cl - secretion participate in theregulation of overall NaCl balance( 41, 42 ). Na readsorption in the IMCD occurs predominantly via apical amiloride-sensitiveNa channels and a basolateral Na -K -ATPase.The activities of other transporters such asNa -K -2Cl - andNa -Cl - cotransporters and theNa /H exchanger may also be involved in Na homeostasis ( 50 ). The IMCDalso has the capacity to secrete anions. The CFTR( 21, 48 ), ClCa channels,swelling-activated Cl - currents( 3, 4, 45 ), and ClC channels( 24 ) have been implicated inthe process. Na absorption and Cl - secretion in the IMCD are controlled by a wide variety of hormones and renal autacoids( 20, 22, 41, 42, 50 ). For instance,mineralocorticoids augment the activities of both apical Na channels and basolateral Na-K -ATPase, whereas atrial natriureticpeptides bind to guanylate cyclase-linked receptors, leading to inhibition ofapical Na channel function( 50 ). Anion secretion can beaffected by changes in intracellular cAMP or Ca 2 ( 4, 21 ).
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The purpose of this study was to characterize in detail the ClCa currentsin IMCD-K2 cells. IMCD-K2 cells were established from the initial portion ofthe IMCD of a mouse transgenic for SV40( 27 ). The cell line retainsfeatures typical of IMCD: cyclic nucleotide-gated cation channels in theapical membrane mediate mineralocorticoid-sensitive Na absorptionthat is inhibited by amiloride( 27 ). Moreover, this cell lineexhibits electrogenic Cl - secretion( 27 ). A recent study presented evidence that mouse IMCD-K2 cells possess two separate apical Cl - conductances that are activated by intracellular cAMP orCa 2  , respectively( 3 ). ClCa currents in IMCD-K2cells showed voltage-dependent kinetic changes in whole cell recording. Afterthe induction of slow ramps in [Ca 2 ] i produced by exposing BAPTA-loaded IMCD-K2 cells to ionomycin, whole cellcurrents exhibited pronounced outward rectification with time dependence. ClCacurrents in another IMCD cell line, IMCD-K3, exhibited somewhat differentproperties ( 40 ) (see DISCUSSION ).
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Our laboratory has studied ClCa channels in Xenopus laevis oocytes extensively ( 18, 28, 29, 31, 32, 38, 39 ). ClCa channels in oocytesresemble Ca 2 -activated Cl - channelsexpressed in some mammalian cells such as parotid acinar cells, lacrimal glandcells, and pancreatic acinar cells( 33 ). They are activated directly by intracellular Ca 2  , with a K d in the 1 µM range, exhibit differentvoltage-dependent kinetics at low and high Ca 2 concentrations ([Ca 2 ]) and possess an anion Cl. However, ClCa channels in other cells aredifferent ( 33 ).Ca 2 activation of ClCa currents in colonic T84 andJurkat T cells is mediated by Ca 2 /calmodulin-regulated protein kinase II, whereas ClCa currents in olfactory receptors have a lowCa 2 affinity, with a K d of 26 µMand an Br. Therefore, itseems that different types of ClCa channels exist in different cell types. Wehave described the "signature properties" of the X.laevis oocyte-type of ClCa channels (see Table 1 in Ref. 33 ). Stated simply,Ca 2 directly activates the channel withoutphosphorylation. The channel has a voltage-dependent Ca 2 affinity (in the 1 µM range) such that, at a[Ca 2 ] i of and are time dependent. However, at high[Ca 2 ], the currents show a linear current-voltage( I - V ) relationship and are time independent. The channel ismore permeant to larger halide Cl). The Cl channel blockers anthracene-9-carboxylic acid (A9C),diphenylamine-2-carboxylic acid (DPC), and DIDS block the channel from theoutside in a voltage-dependent manner.

In this study, we applied whole cell recording to IMCD-K2 cells andobserved Ca 2 activation by directly changing[Ca 2 ] i. Lower Ca 2 ( - currents,whereas high [Ca 2 ] i elicited nearly linearCl - currents. The currents activated at low[Ca 2 ] i were time dependent for activationand deactivation. The Ca 2 activation and the Ca 2 affinity of the channel were voltage dependent. TheCl - channel blocker niflumic acid (NFA) in the bath equallyinhibited both inward and outward currents reversibly, with a K i = 7.6 µM. DIDS, DPC, and A9C reversibly inhibitedoutward currents in a voltage-dependent manner. These biophysical andpharmacological characteristics are so similar to those in X. laevis oocytes that we conclude that the ClCa channels expressed in IMCD-K2 cells andin X. laevis oocytes are very similar.% @% ?0 x6 R  @, ^* v" j, n. O$ P

METHODS

Cell Culture
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Mouse kidney IMCD-K2 cells were kindly provided by Dr. Bruce A. Stanton(Dartmouth Medical School, Hanover, NH), routinely cultured ( passages14-36 ) in PC-1 medium (Bio-Whittaker, Walkersville, MD)supplemented with PC-1 supplement (Bio-Whittaker), 10% heat-inactivated fetalbovine serum (Hyclone, Logan, UT), 2 mM L -glutamine (LifeTechnologies, Grand Island, NY), 50 U/ml penicillin, and 50 µg/mlstreptomycin (Life Technologies) in tissue culture flasks (Costar, Cambridge,MA) coated with Vitrogen plating media containing DMEM (Life Technologies), human fibronectin (10 µg/ml, Collaborative Biomedical, Bedford, MA), 1%Vitrogen 100 (purified collagen; Cohesion, Palo Alto, CA), and BSA (fractionV, 10 µg/ml; Sigma, St. Louis, MO), and maintained in a 37°C and 5%CO 2 incubator. For experiments, cells were seeded onto glasscoverslips (Fisher Scientific, Pittsburgh, PA) and used 1-3 dayslater.

Whole Cell Patch-Clamp Recordings5 D1 e  Q3 O, O  b. i! ?+ @' [

Current recordings from mammalian cells were made using a whole cellconfiguration. Whole cell currents were recorded with borosilicate glasselectrodes (Sutter Instrument), pulled by a Sutter P-2000 puller, and firepolished. Pipette resistances were 2-5 M. Whole cell patch-clampdata were acquired with an Axopatch 200A amplifier controlled by a Clampex 8.1 via a Digidata 1322A analog-to-digital and digital-to-analog converter (AxonInstruments), sampled at 2 kHz, and filtered at 1 kHz with a four-polelow-pass Bessel filter. The bath was grounded via a 3 M KCl-agarose bridgeconnected to an Ag-AgCl reference electrode. Bath solution changes wereperformed with a group of sewer pipes, having an 100-µm internal diameter, connected to the gravity-fed solution containers so that the solution bathingthe cells could be changed in 2 ml/min.
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Solutions

Symmetrical Cl - solutions containing 150 mM Cl wereusually used for the whole cell recordings. Pipette solutions contained (inmM) 150 N -methyl- D -glucamine chloride (NMDG)-Cl, 10HEPES-NMDG (pH 7.3), and 10 EGTA-NMDG or 10Ca 2 -EGTA-NMDG. The bath solution contained (in mM) 140NMDG-Cl, 4.5 KCl, 1 MgCl 2, 2 CaCl 2, and 10 HEPES-NMDG(pH 7.4). Sucrose was used to adjust osmolarity. In some experiments,NMDG was replaced with K in the pipette solution andwith Na in the bath solution. Mg-ATP (2 mM; Sigma) was also addedto the pipette solution.4 c" w7 w$ u' y0 j! J9 r2 S) d

To obtain submicromolar concentrations of free Ca 2  ,we buffered solutions with EGTA using the method of Tsien and Pozzan( 47 ). The stock solution ofCa 2 -EGTA was made by the pH-metric method ( 47 ). Working solutions havingdifferent free Ca 2 were prepared by mixing the0Ca 2 -EGTA solution with theCa 2 -EGTA solution (Molecular Probes) in various ratios.The free [Ca 2 ] was calculated from the equation[Ca 2 ] = K d x [Ca 2 -EGTA]/, where K d is the K d of EGTA ( K d = 1.0 x 10 - 7 M at 24°C, pH 7.3, ionic strength 0.16M). The calculated Ca 2 concentrations were confirmed ineach solution by fura 2 (Molecular Probes) measurements using an LS-50Bluminescence spectrophotometer (PerkinElmer, Norwalk CT).0 }! m& o' u) M# A* @

Anion Channel Blockers& E# o& `6 `* ]2 j# r

NFA, DPC, and tamoxifen were purchased from Sigma, A9C was from AldrichChemical, and DIDS was from Molecular Probes. DIDS was suspended in water at0.3 M as a stock before working solutions were made. Other compounds weredissolved in DMSO at 0.3 M as stocks to keep the DMSO concentration in workingsolutions DL -DTT (Sigma) stock solution was made inH 2 O.

Display and Analysis of Data
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For the calculations and graphical presentation, we used Origin 6.0software (Microcal). Curve fitting was performed using the iterativealgorithms in Origin. Results are presented as means ± SE, and n refers to the number of patches in each experiments. Thesignificance of the difference between values was determined using Student's t -test (paired data) for tests between individual data pairs.' l# d8 }4 o4 ^, d
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RESULTS8 W3 S9 q& M0 R- C1 C+ W
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Activation of Cl - Currents in Whole Cell Patches byCytosolic Ca 2

Figure 1 shows thebiophysical characteristics of ClCa currents in an IMCD-K2 whole cellrecordings. Both extracellular and cytosolic solutions contained NMDG-Cl, sothat cation currents were minimized. The patches were held at -40 mV andstepped to a membrane potential ( V m ) between -100and  100 mV for 0.75 s and then stepped to -40 mV for 0.3 s. At nM Ca 2  , only very small currents were recorded( Fig. 1 A ). However,when [Ca 2 ] was increased to 100 nM, a slowly developing but sustained outward current was observed in response to depolarizing steps( Fig. 1 B ).Deactivating inward tail currents were observed on the return to -40 mVfrom depolarized potentials ( Fig. 1 B ). Outward currents observed with 100-500 nMCa 2 were composed of a small instantaneoustime-independent component and a large slowly activating time-dependentcomponent ( Fig. 1, B - D ). Very little steady-state inwardcurrent was observed at negative potentials at [Ca 2 ] Fig. 1, B and C ). With [Ca 2 ] 500 nM, outwardcurrents were increasingly dominated by the time-independent component andinward currents developed ( Fig. 1, D and E ). At 21 µM Ca 2  , inward and outward currents were nearly equal inamplitude, and the currents became essentially instantaneous( Fig. 1 F ). Figure 1 G shows thatthe steady-state I - V relationship changed from outwardlyrectifying at 2 to almost linear at 21µM [Ca 2 ]. The currents reversed atCl - equilibrium potential. Note that the outward currents at800 nM and 21 µM were smaller than that at 500 nM. We do not know whetherthis was caused by current rundown or whether the channel respondedbiphasically to Ca 2  . We prefer the explanation thatCa 2 has a biphasic effect. A similar phenomenon was observed in excised patches from X. laevis oocytes( 28 ).

Fig. 1. Activation of Cl - currents by intracellularCa 2 in mouse IMCD-K2 cells. A - F : representative traces ofCa 2 -activated Cl - currents recorded inwhole cell configuration with various Ca 2 concentrations ([Ca 2 ]) in pipette solutions. The wholecell patches were voltage clamped by stepping from a holding potential of-40 mV to various potentials between -100 and  100 mV for 0.75 swith a 20-mV increment for each step, followed by a 0.3-s step of -40 mV(voltage protocol is shown in inset in A ). G :steady-state current-voltage ( I - V ) relationship for wholecell currents. The average currents at the end of the 0.75-s pulse fordifferent [Ca 2 ] were plotted vs. membrane potentials( V m ). Nos. of cells are 6, 10, 5, 5, 5, or 9, and SE are31, 79, 69, 193, 317, 351 pA at 100 mV for 2 ], respectively. Error bars are not shownfor clarity.

Similar experiments were done with 150 mM KCl on the intracellular side and150 mM NaCl on the extracellular side. Similar results were obtained exceptthat reversal potentials were shifted from 0 with symmetrical NMDG( Fig. 1 G ) to about-10 mV with K /Na (data not shown), indicatingthat nonselective cation conductance may have contaminated the records under these conditions or that the channel is also slightly permeable for smallcations.9 J& A$ C2 X' _: h% t' D
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Voltage-Dependent Ca 2 Affinity of ClCaCurrents/ z" R9 q" I+ H$ y; ]7 N
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The data in Fig. 1 G indicate that the activation of ClCa currents is voltage dependent at a[Ca 2 ] between 100 and 800 nM. For further analysis, thevoltage dependence of the ClCa currents at different[Ca 2 ] was determined by plotting conductance vs. V m. Conductance was determined by measuring theinstantaneous tail currents (see Fig.1 ) at the beginning of the -40-mV step after various voltagesteps between  100 and -100 mV as shown in Fig. 1 A and dividingby the driving force (-40 mV). Figure2 A shows the voltage dependence of the ClCa conductance at different [Ca 2 ] i. The averageconductance-voltage curves ( n = 5-12 different whole cellpatches for each [Ca 2 ]) show that increasing[Ca 2 ] from 100 to 500 nM shifted the conductance-voltage relationship strongly in the leftward direction andsignificantly increased the voltage dependence (increased the slope),indicating that the activation of the channel by Ca 2 isaffected by V m. We have previously shown that the effect of V m on the Ca 2 activation of X. laevis oocyte ClCa channels is due to the voltage dependence ofthe channel for Ca 2 affinity.4 _# n5 y' @$ {+ H& F- [' ~

Fig. 2. Voltage-dependent Ca 2 affinity ofCa 2 -activated Cl - (ClCa) currents inIMCD-K2 cells. A : voltage dependence ofCa 2 -dependent conductance of whole cell ClCa currents.Average conductance at various [Ca 2 ] was calculated bydividing the tail currents at the beginning of 0.3-s steps (shown in A - F in Fig.1 ) by the driving force (-40 mV). B : averageapparent affinity of the channel for Ca 2 at differentvoltages. The conductance in A was replotted as a function of[Ca 2 ]. The data for,  100mV;, -100 mV. C and D : best-fit parameters ofthe data in B to the Hill equation. n H, Hillcoefficient; EC 50, apparent affinity of the channel forCa 2  .
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To quantify the voltage dependence of the Ca 2 affinity of the channel, we plotted the mean conductance vs.[Ca 2 ] for each voltage( Fig. 2 B ). Only thedata between nM Ca 2 were fitted to theHill equation. Data for 800 nM and 21 µM Ca 2 werenot included when the average data were fitted to the Hill equation, becauseof the rundown of the current at these Ca 2 concentrations. The analysis shows that the apparent affinity of the channelfor Ca 2 (EC 50 ) increased about threefoldfrom 950 nM at -100 mV to 350 nM at  100 mV( Fig. 2 C ). The Hillcoefficient ranged from 2 to 3 from -100 to  100 mV, indicating thatmore than one Ca 2 ion bound to the channel to activateit.

To fit the data to the Hill equation, we calculated a predicted maximumconductance ( G max ) by fitting the conductance data for 100 mV and allowing all the parameters to float. We then assumed that thispredicted G max was the same for all voltages. The data forall voltages were then fit by fixing the G max to thispredicted G max value. This approach assumes that the predicted G max at 21 µM Ca 2 islarger than what is actually recorded. The recorded currents are smallerbecause of rundown or some secondary process. Using this method, theEC 50 is estimated to be 350 nM at 100 mV and 950 nM at -100mV. As an alternative method for estimating EC 50 values, wemeasured the [Ca 2 ] that produced 50% of the observedconductance at 21 µM Ca 2  . Thisconcentration was 169 nM at  100 mV and 550 nM at -100 mV. Therefore,although the EC 50 values calculated by these two methods differ byabout twofold, the conclusion is the same: the EC 50 forCa 2 is smaller at positive potentials than it is atnegative potentials.( @& @  D/ Q: N7 K5 Q* q" b

Pharmacological Properties of ClCa Currents in IMCD-K2 Cells5 m" a. E$ b5 K- p

NFA. Figure 3 showsthe block of ClCa currents by NFA applied to whole cell patched IMCD-K2 cellsin the bath. NFA is one of the most commonly used Cl - channelinhibitors. Cells were patched in the whole cell configuration withsymmetrical NMDG-Cl - solutions applied to intra- andextracellular sides and perfused with bath solutions containing different NFAconcentrations. To observe the inhibition of inward and outward currents byblockers, a high-Ca 2 (21 µMCa 2 ) pipette solution was used to fully activate inwardand outward currents.

Fig. 3. Block of ClCa currents by external niflumic acid (NFA) in IMCD-K2 wholecell recordings. A : I - V relationships showing equalblock of inward and outward ClCa currents by various NFA concentrations() in the bath. The whole cell patch was voltage clamped from the holdingpotential of -40 mV with a 200-ms-duration voltage ramp from -100to  100 mV. Pipette solution contained high [Ca 2 ]. The in the bath solution were 0, 3, 10, 30, and 100-300 µM. B : apparent K i of NFA applied to the bath at V m of  100 mV ( n = 5). The data were fitted tothe equation I / I NFA = 0 = I min ( I max - I min )/{1  (/ K i ) n }, where I max and I min are the maximum andminimum current amplitudes, respectively, K i is theconcentration of NFA required to reduce the current amplitude to( I max I min )/2, and n is theslope factor. C : reversibility of block of ClCa currents by NFA inthe bath. The patched cells were treated with 100 µM NFA in the bath, andthen NFA was washed out. The currents at membrane potentials of -100 and 100 mV were measured and averaged ( n = 5).

Figure 3 A shows I - V relationships recorded in the absence and presence ofvarious concentrations of NFA in the bath. The whole cell patch was voltageclamped from the holding potential of -40 mV with a 200-ms-durationvoltage ramp from -100 to  100 mV. Inward and outward currents werealmost equally blocked in a concentration-dependent manner. The current traces did not show a significant voltage-dependent block. In Fig. 3 B, the averagedcurrents inhibited by each NFA concentration at  100 mV were expressed as afraction of the current at  100 mV in the absence of NFA( I / I NFA = 0) and plotted as a function of NFAconcentration. The data were fitted to the equation of the form
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where I max and I min are the maximumand minimum current amplitudes, respectively, K i is theconcentration of NFA required to reduce the current amplitude to( I max I min )/2, and n is theslope factor. K i at  100 mV for NFA is to 7.6 µM, with n = 1.03. Inhibition at 300 µM NFA is 93% for outward currents at 100 mV. Figure 3 C shows that the averaged inhibition by 100 µM NFA is reversible. NFA was themost potent blocker we tested.- _3 H& M! n% g

DIDS, DPC, and A9C. Three other Cl - channelblockers, namely, DIDS, DPC, and A9C, exhibited a voltage-dependent block. Figure 4 A shows theeffect of DIDS on IMCD ClCa currents. DIDS, unlike NFA, blocks in avoltage-dependent manner. The outward current was blocked at positive voltagesmore than the inward currents at negative voltages. At 300 µM, DIDS blocked outward currents by 80.8% at  100 mV but inward currents by 54.9% at-100 mV ( Fig. 4, D and E ). The block was reversible. DPC behaved in a similarway. It also inhibited ClCa currents in a voltage-dependent manner( Fig. 4 B ). DPC (300µM) blocked outward currents by 70.0% at  100 mV but inward currents by44.4% at -100 mV ( Fig. 4, D and E ). The inhibition was also reversible. A9Creversibly blocked ClCa currents in a voltage-dependent manner( Fig. 4 C ). A9C (300µM) blocked outward currents by 68.3% at  100 mV but inward currents only by 26.8% at -100 mV ( Fig. 4, D and E )./ o# X% F% I3 y3 i# F: c

Fig. 4. A - C : I - V relationships of ClCacurrents blocked by external DIDS, diphenylamine-2-carboxylic acid (DPC), andanthracene-9-carboxylic acid (A9C), respectively, in IMCD-K2 whole cellrecordings. D and E : comparison of reversible block ofoutward ( D ) and inward ( E ) ClCa currents by blockers. DIDS(300 µM, n =5), DPC ( n = 7), or A9C ( n = 7) wasapplied in the bath. For experimental conditions, refer to the legend to Fig. 3.6 b% X( Z& A4 B1 E* a: u  S/ Y0 i( j7 V
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DTT and tamoxifen. DTT, a reducing agent, has been reported tostrongly block heterologously expressed CLCA currents( 10, 14, 16 ). Figure 5 A shows that 2mM DTT has only a slightly inhibitory effect on outward ClCa currents inIMCD-K2 cells. The inhibition developed slowly. DTT (2 mM) inhibited thecurrent by 35% at 100 mV in 4-6 min (Student's t -test, paired data, P = 0.01). A similar result was obtained in X. laevis oocytes (not shown).* ^* o) C9 g% n7 [+ R( J  _1 @
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Fig. 5. A : effect of external DTT on ClCa currents in IMCD-K2 cells.Currents were recorded with step voltages. Voltage protocol refers to Fig.1. The average currents( n = 9 for each) at the end of the 0.75-s pulse were plotted vs.membrane potentials. The patched cells were treated with 2 mM DTT for4-6 min before being washed out. DTT inhibited outward ClCa currents inIMCD-K2 whole cell recordings. Difference significance was determined usingStudent's t -test (paired data, P = 0.01). B : nosignificant block of ClCa currents by external tamoxifen in IMCD-K2 whole cellrecordings. Experimental conditions refer to the legend to Fig. 3. The currents at V m of -100 and  100 mV were measured, averaged( n = 5), and plotted as I - V relationship.
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Tamoxifen is an effective blocker for swelling-activatedCl - channels( 4 ) and CLCA( 17 ), but it did not blockClCa currents after 3-6 min in either IMCD-K2 cells( Fig. 5 B ) or X. laevis oocytes (not shown).

DISCUSSION* Z4 w* k2 b, s9 x* D( t6 Q

Biophysical Properties of ClCa Currents in IMCD Cell Lines# I& u% L6 W- d: l9 ^

ClCa currents have previously been studied in IMCD-3 and IMCD-K2 cell linesderived from IMCD. ClCa currents in IMCD-3 cells ( 40 ) are different from thosein IMCD-K2 cells. In the absence of any agents that would raise intracellularCa 2  , the majority of IMCD-3 cells (64%) possessed alarge, spontaneously active, outwardly rectifying, andtime/voltage-independent Cl conductance ( 45 ). This suggested that theClCa channels were open at resting Ca 2 levels. Changesin [Ca 2 ] i produced by chelating cytosolic Ca 2 by pre-loading the cells with BAPTA-AM or byelevating cytosolic Ca 2 with ionomycin or ATP alteredcurrent amplitude but did not alter the kinetics of the current( 45 ). Because the biophysicalproperties are similar to those described for mouse CLCA1 functionallyexpressed in HEK-293 cells, it was suggested that the ClCa currents in IMCD-3cells might be displayed by the CLCA family( 10 ).
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In contrast, ClCa currents in IMCD-K2 cells( 27 ) showed time and voltagedependence. After the induction of slow ramps in [Ca 2 ] i produced by exposing BAPTA-loadedIMCD-K2 cells to ionomycin, whole cell currents exhibited pronounced outward rectification with time-dependent activation or inactivation ( 3 ). We have extended thesefindings here. We applied whole cell recording to IMCD-K2 cells and observedthe Ca 2 activation by directly changing[Ca 2 ] i. Lower Ca 2 ( outwardly rectifying Cl - currents,whereas high Ca 2 elicited nearly linearCl - currents. The currents activated at lowCa 2 showed time-dependent activation and deactivation. Ca 2 activation and Ca 2 affinityof the ClCa channel were voltage dependent. The Ca 2 activation of the channel was dose dependent, and the EC 50 was 0.35 µM at  100 mV and 0.95 µM at -100 mV. These dataindicate that ClCa currents in IMCD-K2 cells resemble those in secretoryepithelia and X. laevis oocytes( 2, 33 ).  [- U5 b" }; Z

One may be tempted to speculate that the differences in ClCa currentsbetween IMCD-3 and IMCD-K2 cells may be related to the fact that IMCD-3 cellsare derived from the terminal segment of the duct( 40 ) and IMCD-K2 cells arederived from the initial segment( 27 ). However, to ourknowledge, there are no data on Ca 2 -activatedCl - channels in intact IMCD. Therefore, one must consider thepossibility that the ClCa currents in these cell lines may not reflect thesituation in the intact tissue. To determine whether ClCa currents differbetween initial and terminal segments of the IMCD, electrophysiologicalrecording of native cells from different segments of IMCD may clarify thispoint.: z7 p6 y( L2 j  x7 C! R

Comparison of Properties of ClCa Currents Between X. Laevis Oocytesand IMCD-K2 Cells
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We have extensively studied ClCa channels in X. laevis oocytes ( 28, 38, 39 ). Comparing the propertiesof ClCa currents in oocytes with those in IMCD-K2 cells, we found that bothchannels resemble each other biophysically and pharmacologically, as summarized in Table 1. ClCacurrent activation in both cell types was controlled strictly by[Ca 2 ] i, and the Ca 2 activation showed voltage dependency. At low [Ca 2 ] currents activated slowly on depolarization and deactivated on hyperpolarization and the steady-state I - V plot wasstrongly outwardly rectifying. At higher [Ca 2 ], thecurrents did not rectify and were time independent. This difference inbehavior at different [Ca 2 ] was due to the differentaffinity of the channel for Ca 2  . Both ClCa channelsshowed a higher affinity for Ca 2 at positive V m but a lower affinity at negative V m. The affinity difference is about two- to threefoldbetween -100 and  100 mV.
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Table 1. Summary of comparison of ClCa currents in oocytes and IMCD-K2cells1 Y0 L- k" Y0 o$ @1 W
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The similarity of the biophysical properties of ClCa channels for bothIMCD-K2 and oocytes suggests a similar channel pore structure. Thepharmacological properties support the hypothesis. We studied in detail thevoltage-dependent block of ClCa channels by various anion blockers forCl - channels with excised patches from oocytes. All drugsstudied blocked the channel from the outside in a voltage-dependent manner.The order of DPC at  100 mV. Ouranalysis suggests that the channel is an elliptical cone with the largestopening facing the extracellular space( 38 ). In this study, the fourdrugs applied in the bath also mainly blocked the outward ClCa currents inwhole cell recordings of IMCD-K2 cells. The block also showed voltagedependence. The order of potency was similar to that for oocytes, although DIDS was more potent than A9C for IMCD-K2 cells( Table 1 )./ t/ Z' S/ b$ T
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Molecular Identification of Potential Genes for ClCa Currents inIMCD-K2 Cells
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So far, two molecular candidates for ClCa channels have been identified,bestrophins and CLCA. Bestrophin is the product of the gene isolated from aperson with Best vitelliform macular dystrophy (VMD-2) by positional cloning( 35, 37 ). Northern blot analysishas shown that bestrophin mRNA is strongly distributed in retina, brain, andspinal cord, and RT-PCR has indicated that bestrophin also existed in kidney.Bestrophin localizes to the basolateral plasma membrane of the retinal pigmentepithelium (RPE) ( 34 ) andcontributes to the slow light peak of the electrooculogram ( 8 ). The function ofbestrophins has been enigmatic until recently, Sun et al.( 46 ) reported that humanbestrophins are Ca 2 -sensitive Cl - channels. Under whole cell recording, heterologously expressed humanbestrophin-1 in HEK-293 cells showed voltage-independent Cl currents at 40 nMCa 2  . The heterologously expressed human bestrophin-1has very high sensitivity to Ca 2  . The EC 50 is 0.1 µM at  100 mV, and the currents are time independent at all[Ca 2 ] (Qu Z, unpublished observations). Theseproperties are different from those of ClCa currents in IMCD-K2 cells.# U4 _5 x1 i6 x$ F" Y# G
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Although a putative family of Ca 2 -sensitive Clchannels (CLCA) has been cloned( 1, 7, 10, 14 - 17, 43 ), this remains controversial. The properties of the heterologously expressed CLCA channels( 10, 14, 15, 25 ) differ from those ofnative ClCa currents in various cells, but the possibility remains that asubunit is needed for CLCA channels to express currents like those for nativeClCa. Mouse CLCA1 was cloned from a mouse lung library using homology cloning( 10 ). The analysis of the tissue distribution of mCLCA1 showed strong expression in mouse epithelialtissues, including kidney. The properties of mouse CLCA1 have been studied byheterologous expression in HEK-293 cells( 10 ). Mouse CLCA1 currentswere activated by intracellular Ca 2 in whole cellrecording. The currents were outwardly rectifying, but time independent at 2µM [Ca 2 ] i. Obviously, the properties ofmouse CLCA1 currents are not identical to those in IMCD-K2 cells.

Although our RT-PCR data indicated that the bestrophin and CLCA genefamilies were expressed in IMCD-K2 cells (data not shown), bestrophin and CLCAchannels appear to be different from the ClCa currents we have studied inIMCD-K2 cells.# i7 w7 d' K* V) O6 V
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Physiological Functions of ClCa Conductance in IMCD-K2 Cells9 V3 f, h. C8 x" N2 H4 q
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Although the mouse and X. laevis are greatly separated from eachother on the phylogenetic tree, the properties of ClCa channels in bothspecies are very similar, indicating that the channel structure may be highlyconserved; the IMCD-K2 cell line was originally established from the initialIMCD of mice ( 27 ).Reconstituted mouse IMCD-K2 epithelia electrogenically absorb Na ( 12, 27 ) through nucleotide-gatedcation channels and display transepithelial anion secretion( 27 ). Anion secretion inIMCD-K2 cells involves uphill accumulation ofCl - /HCO 3 across the basolateral membrane, followedby downhill movement across the apical membrane through Cl - channels. Boese et al. ( 3 )presented evidence that ClCa conductance was present at the apical membrane ofreconstituted IMCD-K2 epithelial layers and participated in transepithelialanion secretion when ATP and ionomycin were applied to the layers. It islikely that a variety of hormones and paracrines can activate apical ClCacurrents and affect IMCD anion secretion via increasing [Ca 2 ] i.* _3 F9 n: s: t1 {

Wild-type CFTR was also expressed in all segments of rat kidney nephron,including IMCD ( 36 ).Cl - currents mediated by CFTR were also revealed in IMCD andIMCD-K2 cells ( 3, 22, 48 ). Because the renal deficitin cystic fibrosis is not profound compared with that in the pancreas or smallintestine, alternative mechanisms must exist to compensate for the loss ofCFTR function ( 11, 44, 49 ). The possibility existsthat ClCa in mouse IMCD may compensate for defective CFTR. It has beenhypothesized that the severity of the deficit due to defective CFTR indifferent organs correlates with the expression of an apical-epithelial ClCaconductance and that this ClCa conductance can functionally compensate for theloss of CFTR activity in humans as in the transgenic cystic fibrosis mouse( 5, 11, 49 ).3 y6 F' H3 P" P) t

DISCLOSURES
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This study was supported by National Institute of General Medical SciencesGrant GM-60448 (to H. C. Hartzell) and a fellowship from the American HeartAssociation (to Z. Qu).
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