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age, because CAMP mimics the glucagon effect on cell volume. On the other hand, when receptorgated ion channels are activated by hormones, cell volume changes could represent a rather early event in the intracellular signal transduction. Remarkably, the cell volume response to Ca’+-mobilizing hormones is not uniform: phenylephrine swells hepatocytes, whereas extracellular ATl? and vasopressin shrink theml’. This suggests that factors other than [Ca2+li are involved in cell volume changes under the influence of Ca’+-mobilizing hormones. In summary, currently available evidence suggests that changes in cell volume under the influence of hormones act as second or third messengers mediating some of the metabolic hormone effects. It is hoped that this article will stimulate future research in this exciting area. DIETER

HiitJSSINGER* FLORIAN

AND LANG+

‘Medizinische Universitiifsklinik,Hugsfettersfrusse 55, D-7800 Freiburg, Germany. izs;;tutk gshiafhysiologie, Universiliit

References 1 Gammeltoft, S. and van Obberghen, (1986) Biochem. J. 235,1-11

E.

2 Yarden, Y. and Ullrich, A. (1988) Annu. Rev. Biochem. 57,443-478 3 Cohen, P. (1988).Proc. R. Sot. Lond. Ser. B 234,115-144 4 HHussinger, D. and Lang, F. (1991) Biochim. Biophvs. Acta 1071,331-350 5 Jakubowski; J.-and Jakob, A. (1990) Eur. 1. Biochem. 193,541-549 6 Hallbrucker, C., vom Dahl, S., Lang, F., Gerok, W. and H3ussinger, D. (1991) Eur. J. Biochem. 199,467-474 7 Fehlmann, M. and Freychat, P. (1981) J. Biol. Chew 256,7449-7453 8 Chamberlin, M. E. and Strange, K. (1989) Am. 1. Physiol. 257, C159-Cl73 9 Hallbrucker, C., vom Dahl, S., Lang, F., Gerok, W. and Htiussinger, D. (1991) Pfliigers Arch. Physiol. 418, 519-521 10 vom Dabl, S., Hallbrucker, C., Lang, F., Gerok, W. and H;iussinger, D. (1991) Biochem. J. 278,771-777 11 vom Dabl, S., Hallbrucker, C., Lang, F., Gerok, W. and Hlussinger, D. (1991) Biochem. J. 280,105-109 12 Al-Habori, M., Peak, M., Thomas, T. H. and Agius, L. (1992) Biochem. J. 282, 789-796 13 Moule, S. K. and McGivan, J. D. (1990) Biochim. Biophys. Acfu 1031,38>397 14 Hegarty, J. L. et al. (1991) Am. 1. Physiol. 261, C521-C529 Zhang, B. X., 15 Whisenant, N., Khademazad, M., Loessberg, P. and Muallem, S. (1991) Am. J. Physiol. 261, c433-c440 16 Friedmann, N. (1972) Biochim. Biophys.

Acta 274. 214-22.5 17 Moule, S. K. and Mffiivan, J. D. (1990) Eur. 1. Biochem. 187,677-682 18 Mortimore, G. E. and P&ii, A. R. (1987) Annu. Rev. Nuir. 7,539-564 19 Woodside, K. H., Ward, W. F. and Mortimore, G. E. (1974) J. Biol. Chem. 249,5458-5463 20 Baquet, A., Hue, L., Meijer, A. J., van Woerkom, G. M. and Plomp, P. J. A. M. (1990) J. Biol. Chem. 265,955-959 21 Panet, R., Amir, I. and Atlan, H. (1986) Biochim. Biophys. Acta 859,117-121 22 Lang, F. et al. (1992) Pfliiger’s Arch. 420, 42ti27 23 Grinstein, S. and Foskett, J. K. (1990)

Annu. Rev. Physiol. 52,399-$14 24 vom Dal& S., Hallbrucker, C, Lang, F. and Hiussinger, D. (1991) Eur. 1. Biochem. 198, 73-84 25 Baquet, A., Meijer, A. J. and Hue, L. (1991) FEBS Leff. 278,103-106 26 Corasanti, J. G., Gleeson, D., Gautam, A. and Bover, J. L. (1990) Rennl Phvsiol. Biochem. i3,1& . * 27 Suzuki, M. et 01. (1990) Am. J. Physiol. 258, F690-F696 28 Lambert, I. H. (1987) J. Membrane Biof. 98,207-221 29 Sardet, C., Counillon, L., Fran&i, A. and Pouyssegur, J. (1990) Science 247, 723-726

Amiloride:a molecular probe for mechanosensitivechannels Mechanosensitive (MS) membrane ion channels provide a means of transducing membrane deformation or stretch into an electrical or ionic signal. They represent the most recently discovered and least understood of the major channel classes and are the only channel class for which there is no structural or amino acid sequence information (for reviews see Refs l-5). Yet MS channels are ubiquitous, being found in both eukaryotes and prokaryotes3. They are expressed in a wide variety of cell types including both sensory and nonsensary cells4g5. While their role in mechanotransduction in sensory cells is evident, in nonsensory cells they have been implicatedz4 in such diverse functions as cell volume stretchregulation, activated reflexes in vascular endothelium and smooth muscle, gravitaxis and turgor control in and plant cells, cell growth embryogenesis. Although recent evidence indicates there may be a number of classes of MS channels with differing gating and ionselective properties4 as well as different means of conferring mechanosensitivity on a channeP’ the focus here is on the membrane stretch-activated MS cation channel. Experimentally, this channel can be most conveniently activated by applying suction to the patch pipette during cell-attached recordi&. Recently, a controversy has developed concerning the significance of MS channels*. In the extreme view, Morris and Hom9, studying snail neurons, concluded

that MS channels are an artefact of patch-clamp recording, based on their failure to elicit macroscopic MS currents anticipated by singlechannel studies. However, at variance with their result, several groups have now successfully recorded whole-cell macroscopic MS currents in a variety of cell fypes6’1J.11.12_ In particular, Davis et al., demonstrated stretch-activated single-channel and wholecell currents in smooth muscle cells that were longitudinally stretched”. The contradictory results are unexplained but may reflect specific properties of the MS channels and/or cell types studied. One possible and generally overlooked complication2*4*9 in detecting whole-cell MS currents may be related to the recent demonstration that MS channel activity can display rapid (i.e. in less than 0.5 s) and complete adaptation in response to sustained mechanical stimulation”. Furthermore, such adaptive behavior was shown to be fragile to patch recording conditions13. A difficulty in studying MS channels has been the relative lack of pharmacological agents that act seioctively on this class of channel compared with voltageand ligand-gated channels. The existence of selective MS channel blockers would prove highly useful in identifying the physiological role(s) that MS channels play in different nonsensory cells. Furthermore, the availability of high-affinity ligands that bind to MS channels could assist in protein identification and purification procedlures. Although

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-100 mV i7-

Fl

Fig. 7. Characterization of amiloride block of the Xenopus stretch-activated oocyte mechanosensitive channel. a: mechanosetisitive Single channel current events recell-attached corded on patches at - 100 and +50mV with the different external wncentrations. amiloride Notice that amiloride strongly blocks at negative potentials but has no effect at positive potentials. b: Single-channel currenl-voltage relationships measured at different amiloride wncentration. c: Boltzmann distribution describing the voltage-dependence of amiloride block. Note that at extreme negativevoltiges the block becomes voltage-independent reaching a limiting value dependent on amiloride concentration. d: Hill plot for the block measured at - 100 mV, w>kh g&z3 Q ffi! ce’_ efficient of 2. Dashed lines are the theoretical curves for Hill coefficients of I.2 and 3. The solid curves in b. c and d are fits to a theoretical model described in Ref. 17.

+50 @TQ-

0.5 mMamiloride -100 mV

+5Olyph

-w--1.0 mu amiloride +50 e_

-100 mV 7-

4pAI -25 t

240 ms

d

;:p

200

,

,

100

200

-2’ -100

0 WmV)

aminoglycoside antibiotics and various K+ channel blockers, including quinidine, tetraethylammonium and 4-aminopyridine block MS channels, their nonspecificity for this channel class limits their usefulness3. Gadolinium has been shown to be a potent MS channel blocker’* but may also lack channel specific$+. In an effort to find more ideal compounds, we initially became interested in amiloride because it had been shown to block mechanosensitivity in a variety of different sensory systems including hair cells of the ear15. An additional advantage of amiloride for our purposes is that there are over 1000 analogues available for structure-activity studies*6. To extend the study of amiloride blocking action to single MS channels in nonsensory cells we chose the Xenopus oocyte because it expresses a high density of MS channels and is particularly amenable to patch-clamp recording*‘. The oocyte and hair cell MS channels are similar in that both

‘.‘..I

1 1 0.1 [Amiloride] (mM)

exclude anions but are only weakly selective among cations, allowing Naf, K+ and Ca*’ to permeate”*. As illustrated in Fig. 1 we found that external amiloride causes a highly voltagedependent block of the oocyte MS channel in which inward MS channel current was reduced but outward current was almost unaffected17. This voltage-dependent blocking behaviour might arise because the amiloride molecule, which is positively charged at pH 7.2, is driven into the open channel and ‘plugs’ it. However, a number of observations argue against this simple model. First, the block does not increase with hyperpolarization as predicted by a plug model. At extremely negative voltages the relative current approaches a limiting value and becomes voltage independent (Fig. 1). Second, the concentration dependence of the block yields a Hill coefficient of 2 (Fig. 2), inconsistent with a singlesite blocking stoichiometry. Although several models may ex-

plain the above results (see Ref. 17) we favour one involving a voltage-dependent conformational change of the MS channel with subsequent voltage-independent and cooperative binding of two amiloride molecules. Although amiloride does block the oocyte MS channel, the ICs,, of 500 PM (measured at -100 mV) is too high for its use in purification studies. In an attempt to find blockers with higher affinity we screened several amiloride analogues for their blocking action’*. Table I illustrates the blocking potency of amiloride and three analogues on MS currents recorded in oocytes” and mouse hair cells” and on other non-MS ion transport pathwayP. The oocyte sequence matches that reported for the MS current in the mouse hair cell but differs from the analogue blocking sequences of the other transport pathways. The ability to ‘fingerprint’ the amiloride receptor of the MS channel may prove useful in determining its role in specific

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control

mm 0.9 mMamiloride

1 5pA

200 ms

Fig. 2. Amiloride block of stretch-activated mechanosensitive channels recorded in a cell-attached patch from an acutely dissociated toe muscle tibre isolated from a 25dayold dystrophic (mdx)mouse. Suction (20-3OmmHg) was applied by mouth to activate the channels. Both traces were recorded from differentpatcheson the same fibre with a patch potential of appmximatety -90mV. The patch-pipette solution contait?ed 100 rnM fVaCI, 10 rnM HEPES, 10 mM EGTA, pH 7.2, with and withoutamilotide. fWe that the blocking appears to be of similar potency to (or even less potent than) the amitoride block of the oocyte mechanosensitive channel rather than the more potent block of thy hair cell mechanosensitive current.

physiological processes. For example, amiloride has been shown to block cell volume regulation, fertilization and proliferation*O. These all involve an activated increase in Na+ influx through amiloride-sensitive pathways, but the exact pathway involved in each process is unknown*‘. By fingerprinting a particular function according to its block by amiloride analogues, it may be possible to dissect the involvement of MS channels as distinct from the other non-MS ion pathways in that process. Although amiloride and its analogues show a similar sequence in blocking MS channels in the oocyte and MS currents in the hair cell, they are ten times more potent in the latter. We have found that this difference in potency is not due to the different

ionic recording conditions used in the oocyte and hair cell studies. While others have suggested that Ca’+ may be an essential requirement for amiloride block*O, we find that amiloride block in the presence of external Ca*+ (l.SmM) is reduced approximately twofold compared with zero external Ca*‘. Of the amiloride analogues tested on the oocyte, the most potent MS channel blocker was bromohexamethyleneamiloride (BrHMA). This is a photoactivatable compound and could prove useful as a covalently bound label in isolation and purification procedures applied to the MS channel. Photolysis of 6-bromoamiloride analogues has been used previously to identify subunits of the epithelial Na+ channel and the Na+-H+ exchanger16. As with any labelling procedure, care will be

TABLE I. Blockof iontransoorloathwavsbv amilorideand itsanalwues: relativeDOtenCV

Transport pathway MS channel Xenopusoocyte MS channel Hair cell Epithelial Na+ channel Na+-Ca2+ exchanger Na+-H+ exchanger Voltage-gated Na+ channel Voltage-gated Ca + channel

Amiloride (It&,, in vmol)

Benzsmil

1 (500.0)

5.3

1.4

14.7

18

1 (53.0)

8.0

1.3

11.0,

19

1 (0.34)

9.0

co.04

c0.04’

18

1 (1100.0)

11.0

2

11.0’

18

1 (84.0)