Ionic selectivity of volume-sensitivity currents in human epithalial cells

Biochimica et Biophysica Acta, 1139 (1992) 319-323 © 1992 Elsevier Science Publishers B.V. All rights reserved 0925-4439/92/$05.00

319

Rapid Report

BBADIS 60010

Ionic selectivity of volume-sensitive currents in human epithelial cells Andrea Rasola

a,

Luis J.V. G a l i e t t a

a,

D i e t e r C. G r u e n e r t b a n d G i o v a n n i R o m e o "

Laboratorio di Genetica Molecolare, lstituto Giannina Gaslini, Genot,a, (Italia) and b Department of Laboratory Medicine, Cardiovascular and Cancer Research Institute, University of California, San Francisco, CA, (USA) (Received 17 April 1992)

Key words: Chloride current; Cell volume; P-glycoprotein; Epithelial cells; Patch-clamp

The ion selectivity of swelling-activated Cl- currents has been investigated in three different human epithelial cell lines, two derived from the airway epithelium (9HTEo- and CFNPE9o ) and one from a colon carcinoma (T84). The relative permeability of volume-sensitive currents with respect to CI- is: I- (1.19) > NOr (1.07) ~ Br (1.05) > CI (1.0) > F-(0.5) = HCO3(0.48) > isethionate(0.28) > aspartate (0.14) --- gluconate(0.13) ~ SO42 (0.12). This type of ion selectivity is similar to that described for depolarization-activated outwardly rectifying CI channels found in epithelial cells.

Human epithelial cells possess at least three types of CI- currents which are respectively regulated by cAMP, Ca 2+ and cell-volume changes [1,2]. The cAMP-dependent current is related to the expression of the CFTR gene [3]. The identity of Ca 2+- and volume-sensitive currents is instead less defined. It has not been established yet if Ca 2+- and volume-sensitive CI- currents are due to distinct channel types and if they are related to rectifying C1- channels found in patch-clamp studies on epithelial cells [4-6]. It has been recently shown that expression of the P-glycoprotein gene, which shares sequence homology with CFTR, is associated with the presence of a swelling-activated CI- current [7]. Some properties of this current, namely the inactivation at positive membrane potentials and the rectifying current-voltage relationship, resemble those of volume-sensitive C1- currents described in various epithelial cells [8-10]. Nevertheless, other features such as the ion selectivity and the sensitivity to channel blockers, have not been compared. This is required to finally demonstrate that the P-glycoprotein gene encodes for a volume-regulated C1- channel of the same type that is found in airway and intestinal cells. The approach used by Anderson et al. [3], i.e., the comparison of ion selectivity between native and mutated CFTR, may also be applied to the current related to P-glycoprotein. In this study we have determined the permeability

Correspondence to: L.J.V. Galietta, Lab. di Genetica Molecolare, Ist. G. Gaslini, 16148 Genova, Italy.

of volume-sensitive currents toward a wide series of ions. These data will be useful to establish the identity of these currents and their correlation with P-glycoprotein. We have studied 9 H T E o - , C F N P E 9 o - , and T84 cell lines. The former two were obtained from the tracheal epithelium of a normal individual and from the nasal epithelium of a cystic fibrosis patient, respectively [11,12]. Colon carcinoma T84 cells were kindly provided by Dr. J.R. Riordan (Hospital for Sick Children, Toronto). Electrophysiological measurements were carried out using the whole-cell patch-clamp technique [13]. The intracellular (pipette) solution contained (in mM): NaC1 (140), CaC12 (0.18), E G T A (2), MgCl 2 (1), Na2ATP (1), Na-Hepes (10) (pH = 7.3, free Ca 2+= 10 nM) adjusted to 300 m O s m / k g by addition of mannitol. Composition of extracellular solutions was (in raM): NaX (133), CaCI 2 (2), MgCI 2 (2), glucose (10), Na-Hepes (10) (pH = 7.3) where X is C1- (Extracellular Standard Medium) or one of the following anions: I-, Br-, F-, NO 3 , H C O 3 , SO42-, Isethionate, Gluconate or Aspartate. Mannitol was added to obtain the desired osmolality (300 m O s m / k g for isotonic solutions). The hypotonic medium had 90 mM instead of 133 mM NaCl and no Mannitol. The pH of the bicarbonate containing medium was set to pH 7.3 by bubbling CO 2. After preparation, the solution was immediately stored in hermetically closed syringes to avoid loss of CO 2. Throughout the course of experiments this medium was continuously perfused to maintain the H C O 3 concentration constant. Changes of pH were monitored by

320 including Phenol red in media. The F containing solution was prepared without C a 2+ and Mg 2+ to avoid formation of precipitates. A control ESM without Ca 2+ and Mg 2+ was used to check if omission of divalent cations had any effects on chloride currents. Changes of the extracellular solution were made by positioning the perfusion pipette close to the cell. At least 6 - 8 different solutions were tested on the same cell. The reversal potential of m e m b r a n e currents was measured for each ionic condition and relative permeabilities of tested ions with respect to C1 were calculated from Eqn. 1 taken from Gray et al. [14], To minimize liquid junction potentials, the reference electrode was connected to the bath through an agar bridge containing 0.5 M KC1. Potentials which developed at the agar b r i d g e / b a t h junction were measured (0-1 mV with halide ions and 4 - 6 mV with organic anions) and used to correct for the reversal potential of m e m b r a n e currents. Series resistance in our experiments was 4.8___ 2.2 MS'/ (mean_+ S.D.). When analogic compensation of series resistance was not possible due to amplifier oscillations, the uncompensated voltage error was calculated to correct for the applied m e m b r a n e potential. M e m b r a n e potential are given by taking the extracellu-

hypert,

hypot.

!

lar solution as ground. Positive (outward) currents are due to anions flowing into the cell. Experiments were carried out at room temperature (22-25°C). As described previously, whole-cell clectrophysi~logical experiments on T84 [8], 9 H T E o , and CFNPEt;o cells [15] reveal a silent chloride conductance that is activated by the cell swelling after cxposurc to hypotonic solution in the bath. C I current also activates when isotonic bath and pipette solution are used. This phenomenon can be prevented by using a slightly hypertonic (340 m O s m / k g ) extracellular solution [8]. As shown in fig. 1A, the extracellular application of the hypotonic medium causes activation ol: very large currents. Steady-state conductance was reached within 15-20 min after beginning the hypotonic shock and ranged from 3.0-6.0 n S / p F . These large currents caused errors in the applied m e m b r a n e potential, because of the voltage drop resulting from currents flowing through series resistance. To reduce these artifacts, we carried out ion-selectivity studies with isoosmotic bath and pipette solutions (300 m O s m / k g ) . Under these conditions CI conductance spontaneously activates to levels in the range of 0.4-(/.7 n S / p F (Fig. IB). Determination of ion selectivity started as membrane conductance reached a steady-state level. Zero

C

A I

o I

I

2 min.

0.5 nA I /

I

i

1nA l.

L2min"

l

, I

I

Fig. l. Main characteristics of volume-sensitive currents. (A,B), time-course of current activation in hypotonic and isotonic conditions. Traces show membrane currents elicited by stepping the membrane potential to - 100 mV from a holding potential of 0 mV with a frequency of 0.2 Hz. (C,D), superimposed membrane currents evoked by voltage steps in the range - 8 0 to + 80 mV with intervals of 20 mV. These are also shown compressed at the end of trace (B). The holding potential was 0 inV. A conditioning pulse to - 100 mV was applied after the end of the test pulse to reactivate C I - channels which closed during positive steps. Currents in (C) and (D) were obtained with extracellular CI- or gluconate, respectively. Dashed line is the zero current line.

321 current potentials were determined for each ionic condition by direct measurement in current clamp mode (Fig. 2A) or by constructing current-voltage relationships with voltage-clamp measurements (Fig. 2B, C). The first approach was preferentially used (80% of experiments), because many cells showed membrane conductance values too high for voltage-clamp measurements, despite the use of isotonic media. Under these conditions, imposition of membrane potential steps produced flow of large current that can result in artifactual changes of reversal potential due to diffusion polarization. Data obtained by these two approaches overlapped and were pooled. The selectivity of volume-sensitive currents for C1with respect to Na ÷ was determined using a salt gradient created across the plasma membrane. This was accomplished by lowering the extracellular NaCI from

A

133 to 63 mM. As indicated in Table I, a shift of the reversal potentials towards values in the range of + 1 5 / + 17 mV was observed in the three cell lines examined. These values approach the Nernst potential for CI- under these ionic conditions (+ 17.6 mV) and indicate a relative permeability PNa/PcI ranging from 0.02-0.05 (see Table I). The selectivity to various anions was tested. Small changes of reversal potential were produced by substitution of 133 mM extracellular C1- with I-, Br-, and NO 3 . I- showed the highest permeability in all experiments. While NO~- permeability overlapped with that of Br-, both anions were significantly less permeant than I - and more permeant than C1-. Reversal potentials of + 15/+ 17 mV were detected with extracellular F-, indicating a mean relative permeability PF/Pc~ of 0.50.

mV

60

Glucon.

Aspart.

40

S042-

~ t

20 0

-~-

-l,~-~.I--]--~=, _-tNO3"

Br-

--20 I

nA 2

B

- 8 O1

2 rain. I

o

--40

C

- - 8 0I

,/

nA 2

,--40

,

0 mV

oCI-

---1

-2

=NO3~LowCI=Br-

--1

Glucon. I• Isethion. •

--2

Fig. 2. Ionic selectivity of volume-sensitive currents. (A), m e a s u r e m e n t of the zero current potential in the current-clamp mode. Positive and negative deflections of m e m b r a n e potential are the effect of replacing 133 m M extracellular C I - with an equivalent concentration of another anion. Low C1 represents an extracellular NaC1 concentration of 63 mM. (B,C), current-voltage relationships derived from a different experiment in the voltage-clamp mode. Data were obtained with the voltage stimulation protocol described in Fig. 1C and D.

322 The permeability of volume-sensitive channels to other polyatomic anions was also tested. Bicarbonate permeability appears to be about half of that of C 1 . This suggests that under physiological conditions volume-sensitive CI- channels may be involved in bicarbonate transport and intracellular pH regulation. This is consistent with the finding in MDCK cells that a cytoplasmic acidification during hypotonic shock is due to the loss of bicarbonate through anion channels [16]. It is also apparent that other large anions have low, but not negligible, relative permeabilities (Table I). For example, with extracellular sulphate, gluconate, or aspartate, the zero current potential lies in the range + 3 5 / + 45 mV. These values are significantly less positive than the theoretical Nernst potential for C1( + 7 2 mV). From the data shown in Table I, the following selectivity series can be calculated (mean relative permeabilities Px/Pc~ in parentheses):

TABLE 1 Relatit'e permeabilio~ of et'eo" ion and cell lira' t~'~'ted with respect r~, CI Relative permeability with respect to CI is reported for each km and cell line. The mean reversal potential (in mV) is shown in p a r e n t h e s e s + t h e standard deviation. Each mean value was derived from at least 6 experiments. The permeability to Na ~ was measured with a salt gradient as described in the text. The relative permeability' of each ion did not shown significant differences among cell lines (P>> 0.05). Anions showing similar permeabilities (P ~ 0./151 arc grouped. Ion

I

NOr t

CI

I-(1.19)>NO;(1.07)=Br-(1.05)>CI (1.00) > F - (0.50) = HCO~- (0.48)

HCO3

> isethionate(0.28) > aspartate(0.14)

Isethionate

~- gluconate(0.13) = SO 2 (0.12)

Aspartate '

The selectivity sequence obtained from our experiments does not show differences among cell lines, thus, demonstrating that cell swelling activates an homogeneous CI- conductance in epithelial cells of various origin. The halide selectivity, I - > Br > CI > F - , is identical to that of outwardly rectifying CI- channels found in Necturus enterocytes [17], colonic T84 cells [6], and airway epithelial cells [18]. The order of selectivity corresponds to the first Eisenman's sequence and suggests that permeant anions interact with weak cationic sites in the channel. The halide selectivity therefore derives mainly from differences in dehydration energy [19]. A significantly different selectivity has recently been described for cAMP-dependent C1- channels that appear in HeLa and NIH 3T3 cells transfected with the cystic fibrosis transmembrane conductance regulator (CFTR) gene [3]. These channels have a permeability sequence B r - >_ CI - > I - > F-. Less information is available on large anions. In our study we have found that bicarbonate is about half as permeable as CI-, a result matching that obtained by Kunzelmann et al. [20] for outwardly rectifying CIchannels. Sulphate ions, often used as impermeant anions in C1- channels studies, were able to carry currents through volume sensitive channels. It should be noted that glueonate appeared not to enter through cAMP-dependent CI- channels of human airway epithelium [21], thick ascending limb of the mouse kidney

Cell line T84

Gluconate Sulphate Na +

1.19 (-4.4_+1.5) 1.117 ( 1.8+1.01 1.04 ( I.I+0.41 1.0 (0.4 f 0.7l 0.51 (15.4_+ 1 . 0 ) 0.45 118.1_+2.4) 0.26 (29.9_+4.7) 0.16 (39.3_+4.5) 0.15 (40.9_+3.6) 0.12 137.1+2.4) 0.05 (15.5+1.2)

9HTEo 1.19 4.4+11.7l 1.(17 (-I.9_+0.6) 1.(}5 (--1.4+0.41 I.(i (11.2 + 11.4) 0.48 (16.9+0.8) tt.51t (15.9+1.2) 0.30 (27.0 + 1.41 0.12 (44.4+2.2) 0.12 143.9 -+4.5) 0,12 135.6+_2.1) 0.04 (15.8 +(l.7) (

CFNPE9o 1.18 4.2+0.5) 1.111~ l- 1.6+11.41 1.07 ( 1.8 +0.5) 1.0 (0. I + 0.71 0.52 (14.tJ + 1.81 0.4t~ ( 10.2 -+l).SI 0.27 (29.0 + 2.2l i).14 142.3 + 2.7l ~).11 (45.4+2.0) 11.12 (35.6+2.91 0.02 ( 16.(~+ 1.7)

i

[22], and T84 cells (Galietta et al., data not shown). The permeability to gluconate suggests that volumesensitive channels, unlike those activated by cAMP, have a pore diameter larger than 5.4 A. Our data show strong similarities between selectivity properties of volume-sensitive currents and those described for depolarization-activated rectifying CIchannels. It will be interesting to compare these data with those eventually obtained with P-glycoprotein associated currents. Valverde et al. [7] have only tested gluconate ions obtaining a Pgl . . . . . t e / P c l of 0.15. Until now P-glycoprotein has been considered as an ATP-dependent membrane transporter which pumps various drugs out of the cell, thus, allowing tumoral cells to survive to chemioterapic treatments [23]. The C1- channel activity of P-glycoprotein is therefore difficult to be reconciled with the multidrug resistance phenotype. Volume-sensitive CI- currents represent the largest conductive component of the plasma membrane of epithelial cells such as those studied in this paper. If they are due to P-glycoprotein activity, it has to be

323

explained why they are so highly expressed in ceils which have not been selected for drug resistance. This work was supported by NIH grant DK39619 and CF Foundation grant RO 149 (to D.C. Gruenert) and by Italian National Research Council (CNR) grants from the Progetto Finalizzato Biotecnologie e Biostrumentazione (to Prof. Luigi De Cecco). References 1 Cliff, W.H. and Frizzell, R.A. 11990) Proc. Natl. Acad. Sci. USA 87, 4956-4960. 2 Wagner, J.A., Cozens, A.L., Schulman, H., Gruenert, D.C., Stryer, L. and Gardner, P. (1991) Nature 349, 793-796. 3 Anderson, M.P., Gregory, R.J., Thompson, S,, Souza, D.W., Paul, S., Mulligan, R.C., Smith, A.E. and Welsh, M.J. (1991) Science 253, 202-205. 4 Welsh, M.J. and Liedtke, C.M. 11986) Nature 322, 467-470. 5 Frizzell, R.A., Rechkemmer, G. and Shoemaker, R.L. (1986) Science 233, 558-560. 6 Halm, D.R,, Rechkemmer, G.R., Schoumacher, R.A. and Frizzell, R.A. (1988)Am. J. Physiol. 254, C505-C511. 7 Valverde, M.A., Diaz, M., Sepulveda, F.V., Gill, D.R., Hyde, S.C. and Higgins, C.F. (1992) Nature 355, 830-833. 8 Worrell, R.T., Butt, A.G, Cliff, W.H. and Frizzell, R.A. (1989) Am. J. Physiol. 256, Cl111-C1119.

9 McCann, J.D., Li, M. and Welsh, M.J. (1989) J. Gen. Physiol. 94, 11)15-1036. 10 Solc, C.K. and Wine, J.J. (1991) Am. J. Physiol. 261, C658-C674. 11 Gruenert, D.C., Basbaum, C.B., Welsh, M.J., Li, M., Finkbeiner, W.E. and Nadel, J.A. (1988) Proc. Natl. Acad. Sci. USA 85, 5951-5955. 12 Cozens, A.L., Yezzi, M.J., Chin, L., Finkbeiner, W.E., Wagner, J.A. and Gruenert, D.C. (1992) Proc. Natl. Acad. Sci. USA (in press). 13 Hamill, O.P., Marty, A., Neher, E., Sakmann, B. and Sigworth, F.J. (1981) Pfluegers Arch. 391, 85-100. 14 Gray, P.T.A., Bevan, S. and Ritchie, J.M. (1984) Proc. R. Soc. Lond. B221, 395-409. 15 Galietta, L.J.V., Barone, V., Gruenert, D.C. and Romeo, G. (1991) Adv. Exp. Med. Biol. 290, 3//7-317. 16 Ritter, M., Paulmichl, M. and Lang, F. (1991) Pfluegers Arch. 418, 35-39. 17 Giraldez, F., Murray, K.J., Sepulveda, F.V. and Sheppard, D.N. (1989) J. Physiol. 416, 517-537. 18 Frizzell, R.A. (1987) Trends Neurosci. 10, 190-193. 19 Wright, E.M. and Diamond, J.M. (1977) Physiol. Rev. 57, 109-186. 20 Kunzelmann, K., Gerlach, L., Frobe, U. and Greger, R. 11991) Pfluegers Arch. 417, 616-621. 21 Duszyk, M., French, A.S. and Paul Man, S.F. (1990) Biophys. J. 58, 223-230. 22 Paulais, M. and Teulon, J. (1990) J. Membr. Biol. 113, 253-260. 23 Endicott, J.A. and Ling, V. (1989) Annu. Rev. Biochem. 58, 137-171.