Microvillar Ion Channels: Cytoskeletal Modulation of Ion Fluxes

J. theor. Biol. (2000) 206, 561}584 doi:10.1006/jtbi.2000.2146, available online at http://www.idealibrary.com on

Microvillar Ion Channels: Cytoskeletal Modulation of Ion Fluxes KLAUS LANGE Kladower Damm 25b, 14089 Berlin, Germany (Received on 6 March 2000, Accepted in revised form on 17 July 2000)

The recently presented theory of microvillar Ca> signaling [Lange, K. (1999) J. Cell. Physiol. 180, 19}35], combined with Manning's theory of &&condensed counterions'' in linear polyelectrolytes [Manning, G. S. (1969). J. Chem. Phys. 51, 924}931] and the "nding of cable-like ion conductance in actin "laments [Lin, E. C. & Cantiello, H. F. (1993). Biophys. J. 65, 1371}1378], allows a systematic interpretation of the role of the actin cytoskeleton in ion channel regulation. Ion conduction through actin "lament bundles of microvilli exhibits unique nonlinear transmission properties some of which closely resemble that of electronic semiconductors: (1) bundles of micro"laments display signi"cant resistance to cation conduction and (2) this resistance is decreased by supply of additional energy either as thermal, mechanical or electromagnetic "eld energy. Other transmission properties, however, are unique for ionic conduction in polyelectrolytes. (1) Current pulses injected into the "laments were transformed into oscillating currents or even into several discrete charge pulses closely resembling that of single-channel recordings. Discontinuous transmission is due to the existence of counterion clouds along the "xed anionic charge centers of the polymer, each acting as an &&ionic capacitor''. (2) The conductivity of linear polyelectrolytes strongly decreases with the charge number of the counterions; thus, Ca> and Mg> are e!ective modulator of charge transfer through linear polyelectrolytes. Field-dependent formation of divalent cation plugs on either side of the microvillar conduction line may generate the characteristic gating behavior of cation channels. (3) Mechanical movement of actin "lament bundles, e.g. bending of hair cell microvilli, generates charge translocations along the "lament structure (mechano-electrical coupling). (4) Energy of external "elds, by inducing molecular dipoles within the polyelectrolyte matrix, can be transformed into mechanical movement of the system (electromechanical coupling). Because ionic transmission through linear polyelectrolytes is very slow compared with electronic conduction, only low-frequency electromagnetic "elds can interact with the condensed counterion systems of linear polyelectrolytes. The delineated characteristics of microvillar ion conduction are strongly supported by the phenomenon of electro-mechanical coupling (reverse transduction) in microvilli of the audioreceptor (hair) cells and the recently reported dynamics of Ca> signaling in microvilli of audio- and photoreceptor cells. Due to the cell-speci"c expression of di!erent types and combinations of ion channels and transporters in the microvillar tip membrane of di!erentiated cells, the functional properties of this cell surface organelle are highly variable serving a multitude of di!erent cellular functions including receptor-mediated e!ects such as Ca> signaling, regulation of glucose and amino acid transport, as well as modulation of membrane potential. Even mechanical channel activation involved in cell volume regulation can be deduced from the systematic properties of the microvillar channel concept. In addition, the speci"c ion conduction properties of micro"laments combined with their proposed role in Ca> signaling make microvilli the most likely cellular site for the interaction with external electric and magnetic "elds.  2000 Academic Press E-mail: [email protected] 0022}5193/00/200561#24 $35.00/0

 2000 Academic Press

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1. Introduction In the past years a large number of studies have demonstrated the involvement of the actin cytoskeleton in ion channel regulation; however, the mechanism by which actin "laments regulate ion #uxes through plasma membrane channels remained obscure. As recently reviewed by Janmey, 1998, various cation channels including epithelial Na> channels, K> channels, K> (ATP) channels, as well as several cation exchangers and anion channels are modulated by actin e!ectors or by direct interaction with G- or F-actin. Similarly, the mechanism of stretch and volume activation of ion channels is known to depend critically on intact actin cytoskeleton, but again, a productive mechanistic idea of mechano-activation is still missing. The recently proposed notion of microvillar Ca> signaling (Lange, 1999) opens novel aspects on the problem of ion channel/cytoskeleton interaction and may lead to an improved comprehension of the involvement of the actin cytoskeleton in the regulation of transporter and channel activity in di!erentiated cells. A mechanistic concept can now be presented in which fundamental channel properties and functions can be systematically deduced from the properties of only one single biochemical structure, the actinbased cytoskeleton of microvilli. The microvillar concept comprises a variety of di!erent channel types such as receptor-operated, stretch- and volume-activated channels, ATP-dependent K> channels, Ca>-activated channels, ion exchangers, as well as mechanosensitive channels of the hair cell type. This hypothesis connects several "elds of cellular physiology by a consistent unifying theoretical concept. 2. The concept of microvillar signaling 2.1. SPECIAL SURFACE ORGANIZATION OF DIFFERENTIATED CELLS: MICROVILLAR CELL

FIG. 1. Schematic presentation of the microvillar structure. Functional membrane proteins are localized at the tips of microvilli. In the unstimulated state, the cytoskeletal di!usion barrier of actin "laments blocks the di!usion of ions and substrates from the tip compartment to the cytoplasm.

#uxes via anion and cation channels (Lange, 1999; Lange et al., 1989, 1990, 1996, 1997, 1998; Lange & Brandt, 1990a, b, 1993a, b, 1996; Gartzke & Lange, 1996, Gartzke et al., 1997). This type of regulation of metabolism and cell functions appears to be characteristic for di!erentiated cells. According to this notion, functionally important integral membrane proteins including glucose transporters and ion channels are localized within special surface organelles, called microvilli. The tip compartments of these organelles form a multitude of small pericellular spaces on the cell surface, termed entrance compartment, into which hexoses (Lange et al., 1989, 1990; Lange & Brandt, 1990a, b) and ions (Lange & Brandt, 1993a, b; Lange et al., 1996, 1997) can be taken up without restriction. The entrance compartment is, however, separated from the cytoplasm by a tightly aligned bundle of actin "laments representing an e!ective di!usion barrier (Fig. 1). 2.2. DIFFERENTIATION-DEPENDENT SEGREGATION OF MEMBRANE PROTEINS TO MICROVILLI TIPS

THE DIFFERENTIATED STATE

2.2.1. Surface organization of undi+erentiated cells

Recent work on cellular regulation via specialized cell surface organelles, called microvilli, has yielded evidence for a common mechanism regulating various forms of transport and uptake processes such as glucose transport and ion

In undi!erentiated, rapidly growing (embryonic or tumor) cells, functionally important membrane proteins as well as membrane lipids were continuously supplied to the plasma membrane by exocytic events [Fig. 2(a)]. Exocytic

SURFACES, THE MORPHOLOGICAL CORRELATE OF

MICROVILLAR ION CHANNELS

FIG. 2(a). Exocytic insertion of lipid vesicles into the plasma membrane of undi!erentiated cells. Exocytosis is followed by integration of the inserted membrane domain and lateral di!usion of integral membrane proteins.

membrane vesicles, originating from the endoplasmic reticulum, were inserted into the cell surface by fusion with the plasma membrane and, by turning inside out (blebbing), they form small spherical surface protrusions. Subsequently, the inserted membrane domain becomes integrated into the plasma membrane and the included integral membrane proteins distribute over the whole cell surface by lateral di!usion [Fig. 2(a)]. By this mechanism, a great number of integral membrane proteins, essential for cell growth and metabolism, were recruited to the surface of rapidly dividing cells. Under these conditions, maximal activity of life-preserving membrane functions such as glucose and aminoacid transport, regulation of ionic in- and e%uxes is warranted. Uptake rates are exclusively determined by metabolic demands and enzymatic limitations (metabolic control). 2.2.2. Surface organization of di+erentiated cells Under conditions of restricted growth or during cell di!erentiation, generally induced by depletion of metabolic substrates, serum factors, or high cell density (con#uence), the events following exocytosis proceed in a di!erent way. As shown in Fig. 2(b), exocytic vesicles are not integrated into the plasma membrane. Instead, a surface coat of proteoglycans (Lories et al., 1992; Kato et al., 1995; Leppae et al., 1992.) due to its transmembrane organization stabilizes the exocytic membrane domain and inhibits lateral di!usion of integral membrane proteins (Lange & Brandt, 1990b). Subsequent nucleation and growth of actin "laments at this membrane site,

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FIG. 2(b). Mechanism of microvillus formation on di!erentiated cells. Exocytic membrane domains remain stabilized on the cell surface. Transmembrane proteoglycans inhibit lateral di!usion of integral membrane proteins. Microvilli are formed by nucleation and growth of actin "laments at the cytoplasmic surface of these membrane sites.

causes the newly inserted membrane patch to grow out from the cell surface forming a microvillus [Fig. 2(b)]. Microvilli-covered surfaces are characteristic for cells during growth arrest at the G0/G1 phases of the cell cycle or in the di!erentiated state (Tilney & Tilney, 1992; Friedrich et al., 1989; Drenckhahn & Dermitzel, 1988; Boman et al., 1983). Under these conditions, synthesized functional proteins are exclusively translocated to microvilli tips and are thus no longer able to support maximal rates of cellular metabolism. The cytoskeletal di!usion barrier of the microvillar shaft now attenuates the activity of transporters and channels. The metabolic state resulting from this type of surface organization has been termed membrane- or transport-limited (Elbrink & Bihler, 1975). The preferred or exclusive localization of various types of ion channels including non-selective cation, Na>, K> and anion channels on microvilli is documented for a multitude of di!erent cell types. Most likely, stable segregation of membrane proteins into microvilli a!ords direct or indirect binding of their cytoplasmic domains to microvillar actin "laments. Otherwise, un"xed membrane proteins would leave the microvillar membrane domain during transient stimulation. The central role of speci"c linker proteins, e.g. the ezrin/radixin/moetin (ERM) proteins, in the formation and maintenance of the microvillar surface organization has recently been demonstrated in cultured "broblasts and A431 cells by Yonemura & Tsukita 1999, as well as Oshiro

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et al. 1999. Various receptor-regulated e!ector systems, known to be localized to microvilli including glucose transporters and ion channels are able to interact with the actin cytoskeleton via speci"c linker proteins including K> channels (Jing et al., 1997), epithelial Na> channels (Smith et al., 1991; Zuckerman et al., 1999) the erythrocyte anion channel (Schwaetz et al., 1997) CFTR (Short et al., 1998), the insulin-sensitive glucose transporter GluT4 (Kao et al., 1999), the Na>/K>-ATPase (Devarajan et al., 1994), and the Na>/H exchanger (Ediger et al., 1999). As it appears, a speci"c group of functional proteins is selected to reside exclusively within microvillar membranes because of their stable binding to cortical actin "laments, whereas other proteins lacking binding sequences for linker proteins, may have limited residence times within microvilli. 2.3. FUNCTIONAL SIGNIFICANCE OF THE MICROVILLAR SURFACE ORGANIZATION

The localization of functionally important integral proteins on microvilli tips is a new, hitherto unknown aspect of cellular di!erentiation (Lange & Brandt, 1990b). This type of surface organization implies a number of intriguing consequences that are essentially due to sealing of the cell surface preventing unrestricted in#ux of metabolic substrates and ions. Most importantly, cell functions become subject to regulation by external signals including hormones and Ca>. 2.3.1. Regulation of energy metabolism in di+erentiated cells One of the consequences of the microvillar surface organization is the functional integration of the highly e$cient oxidative ATP production into the general framework of glycolytic metabolism. Reduction of glycolysis to rates just matching the demand of the Krebs cycle for C3 substrates enables mitochondria to take over the main energy production of the cell. The characteristic organization of energy metabolism in di!erentiated cells, known as transport-limited metabolism, greatly depends on the microvilar mechanism of glucose transport regulation. The length of the microvillar shaft region, i.e. the e!ectiveness of the di!usion barrier, is regulated by the cytoplasmic

ATP/ADP system in such a manner that glucose uptake is inversely coupled to the availability of cytoplasmic ATP (Lange et al., 1989, 1990). By this mechanism, glucose uptake is precisely regulated and maintained at a level necessary for the production of su$cient ATP via the oxidative pathway. As shown for cultured adipocytes (Lange et al., 1990) and other cells (Shepard et al., 1993, 1997; Shepard & Park, 1994), a relevant increase of external glucose concentration is followed by a conspicuous increase in the length of microvilli. Microvilli elongation, i.e. enhancement of the di!usion barrier function, is supposed to be the mechanism of the well-known phenomenon of glucose-induced transport reduction (&&glucose curb'') (Lange et al., 1990). An important consequence of the establishment of this type of metabolic regulation is the dramatically reduced concentration of cytoplasmic-free glucose. Whereas cell systems with metabolic regulation such as erythrocytes, tumor cells, embryonic cells, and rapidly growing cells in culture exhibit high cytosolic glucose levels reach ing that of the external medium, the cytoplasm of membrane-limited systems are almost free of glucose (Lange et al., 1989, 1990). Restricted in#ux and low cytoplasmic glucose concentrations are experimental criteria for membranelimited systems (Elbrink & Bihler, 1975). 2.3.2. ¹he microvillar di+usion barrier, a precondition for Ca> signaling in di+erentiated cells Another important consequence of the microvillar surface organization in di!erentiated cells is the e!ective in#ux restriction for divalent cations and the establishment of a high-a$nity storage system for cytoplasmic-free Ca> (Lange & Brandt, 1996) that are preconditions for the function of cellular Ca> signaling, the most important signal system of di!erentiated cells. The maintenance of the steep [Ca>] gradient between the extra- and intracellular space is one of the essentials for regulation of cellular functions by cytosolic Ca>. The cytoskeletal system of microvilli provides for both an e!ective blockade of cation entry into the cell and the high-a$nity Ca> storage system to maintain the low cytoplasmic [Ca>].

MICROVILLAR ION CHANNELS

2.3.3. Activation of ion -uxes through microvilli A recently proposed mechanism of cellular Ca> signaling (reviewed in Lange, 1999), essentially depends on the dual function of the microvillar F-actin system acting both as a di!usion barrier and as a high-a$nity Ca> store (Lange, 1999; Lange & Brandt, 1993a, b, 1996; Lange et al., 1996, 1997). Receptor-mediated F-actin dissociation/reorganization of the microvillar actin "laments at the same time releases Ca> from the F-actin store and opens the in#ux pathway for external Ca> into the cell. This double function of F-actin e!ectively couples Ca> release to the in#ux pathway for external Ca> (Fig. 3). Thus, essential cellular functions such as contraction, secretion, modulation of membrane potential, and cell growth are stimulated via the phospholipase C-coupled Ca> signal pathway by depolymerization and reorganization of actin "laments (Lassing & Lindberg, 1985; Goldschmidt-Clermont et al., 1990, 1991). A similar signaling mechanism was suggested for the insulin action on ion and substrate transport. Insulininduced activation of PI 3-kinase initiates reorganization of the cellular actin cytoskeleton and impairs the microvillar di!usion barrier in a similar manner (Lange et al., 1998). In addition to receptor-mediated signaling, the e!ectiveness of the cytoskeletal di!usion barrier can be modulated by several other mechanisms: E

Mechanical forces generated during hyposmotic cell swelling shorten microvilli.

FIG. 3. Activation of Ca> in#ux via the entrance compartment at the microvilli tips occurs by receptor-mediated reorganization of the microvillar F-actin di!usion barrier allowing Ca> entry into the cytoplasm (Lange & Brandt 1996).

E

E

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Exposure to lipophilic compounds alters physical properties of the plasma membrane (Lange et al., 1996; Gartzke et al., 1997), giving rise to dissociation of the bindings between membrane and cytoskeleton in microvilli. This type of interaction of xenobiotics,including thapsigargin (Lange & Brandt, 1993a; Lange et al., 1997; Gartzke et al., 1997) and phorbol esters (data not published), with the cell surface is characterized by typical shape changes of microvilli indicative for an activated Ca> in#ux pathway by impairment of the cytoskeleton}membrane interaction (Gartzke & Lange, 1996; Gartzke et al., 1997). Subtle mechanical load such as small bending of hair cell microvilli induced by acoustic waves or by streaming blood on microvilli of vascular endothelial cell activates the microvillar ion conduction pathway. 3. Electrical properties of actin 5laments

The observed structural organization of ion channels within microvilli strongly suggests that the ion conduction properties of F-actin may govern at least some aspects of ion channel behavior. Micro"laments that are enwrapped in a cable-like manner with an isolating lipid membrane represent cellular organnelles with unique electrical properties. 3.1. F-ACTIN AS DIFFUSION BARRIER FOR IONS

As pointed out above, the formation of microvilli on the surface of di!erentiated cells may be considered as a physiological arrangement for sealing the surface of di!erentiated cells against Ca> in#ux via membrane channels and to establish the intracellular ionic conditions necessary for Ca> signaling. The barrier function of the microvillar actin "lament bundle rests on its structure of a dense matrix of linear polyelectrolytes closely resembling a cation exchanger (Fig. 4). The high density of "xed positive charges with F-actin bundles imparts to this structure considerable resistance to ionic transduction. Whereas anions are largely excluded from the negative matrix of actin "lament bundles, cation conductivity depends on their charge number and on supplied external energy of either

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microvillar pathway must be accompanied by a counter#ux of other cations. Hence, the microvillar pathway may have a cation exchanger function for those internal and external ions that are able to pass both the tip channels and the cytoskeletal matrix. Cation exchangers of this type are well known in cell physiology: Na>/H>, K>/H>, and Na>/K>. The other known exchanger for Na>/Ca> cannot be active in the tight bundle structure of the unstimulated microvillus but a!ords a preceding activation. FIG. 4. Actin "lament bundles within microvilli represent polyelectrolytes acting like cation exchangers. Association of counterions to "xed charges on the polymer increases the resistance for ionic conduction. Accordingly, cell types with microvillar surfaces (epithelial cells) exhibit reduced membrane potentials. Due to their tighter binding to the "xed charges, conduction of divalent cations is largely inhibited.

electrochemical or thermal origin. High in#ux resistance for divalent cations enables di!erentiated cells to maintain the extremely steep Ca> gradient of 10-4 between the intra- and extracellular space and to generate the rapid Ca> increases following receptor stimulation. In contrast to divalent cations, the monovalent cations Na> and K> can pass micro"lament bundles with moderate resistance (Lin & Cantiello, 1993). As an important consequence of the microvillar resistance even for monovalent cation transduction, di!erentiated cells with microvilli-covered surfaces exhibit much lower transmembrane potentials than expected (Fig. 4) (Greenwood et al., 1993). Hepatocytes, for instance, display membrane potentials of !30 mV, which can be signi"cantly enhanced by cell swelling or receptor-mediated activation (Wang & Wondergem, 1993a, b) (see Section 3.5.3). 3.2. THE MICROVILLAR DIFFUSION BARRIER ACTS AS ION EXCHANGER

The anionic matrix of tightly aligned actin "lament bundles in microvilli largely excludes soluble anions. Anion conduction can only be expected within more loosely organized structures such as "lament networks. However, when anion conduction through the microvillar pathway is largely abolished, electroneutrality postulates that all cation movements via the

3.3. MECHANISM OF ION CONDUCTION ALONG ACTIN FILAMENTS

Fixed anionic charges within the matrix of F-actin bundles are neutralized by cationic counter ions in the immediate vicinity of the charge centers (Fig. 5). The spatially coordinated arrangement of counter ions corresponds to a moderate binding of these cations to the "xed charge center. Consequently, free ionic movement along an electric "eld is restricted. Transport of cations along a linear matrix of "xed charges requires the simultaneous movement of counterions from one "xed charge center to the next along the whole length of the conducting path. Simultaneous jumping events, however, afford that all moving ions along the whole conduction path must be provided with the necessary activation energy at the same time. According to the Boltzmann equation of statistical mechanics, the probability, that an ion has the extra energy (E ) needed for translation to the ? next cationic binding site, is proportional to exp (!E /R¹ ). Simultaneous activation of a number ? (n) of such events a!ords very much higher total activation energy. The probability to reach this state is proportional to [exp(!E /R¹ )]L, i.e. ? exponentially decreasing with the length of the conducting pathway. As shown in the following section, the picture of single counterions is a simpli"cation that does not account for all conductance properties of micro"lament bundles. Especially, the principle of simultaneous jumping is an inappropriate picture because each "xed charge on the polymer is opposed by a cloud of counterions. Thus, single ions may change from one cloud to the next giving rise to locally restricted excess of partial

MICROVILLAR ION CHANNELS

FIG. 5. The &&hopping model'' of electrodi!usion of ions along linear polyelectrolytes induced by application of external thermal or "eld energy. Conduction of cations along the polycentric matrix of "xed charges occur by simultaneous jumping of cations to the next charge centers along the whole length of the "lament. Conduction by the &&simultaneous-jump'' mechanism a!ords high activation energies.

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FIG. 6. Schematic presentation of Manning's theory of condensed counterions in linear polyelectrolytes (Manning, 1996). At high spatial density of "xed charge along the polymer, the thermal energy of counterions is lower than the energy necessary to dissociate into the external medium (E ), but higher than E , the energy for cation transfer to the   next charge center.

charges along the conducting pathway. An experiment of Lin and Cantiello impressively demonstrated the unique electrical features of this type of ion conduction. 3.4. CABLE-LIKE CONDUCTANCE IN F-ACTIN BUNDLES

In a recent study, Lin & Cantiello (1993), demonstrated cable-like conductance along F-actin "laments in an aqueous environment. Cable-like conductance is de"ned as the property of extended polymers to conduct ionic currents better along the "lament axis than through the surrounding salt solution. The theory of this phenomenon is based on the hypothesis of &&condensed counterions'' as proposed by Manning (1969) (Fig. 6). In linear polymers with high charge density, cable-like conductance occurs when the thermal energy of the counterions is identical or larger than the energy necessary for its transfer to the next charge center, but lower than the dissociation energy into the free solution (Fig. 5: E 'k¹'E ). Under these conditions, ions   can move along the polymer "lament, but are hindered to leave the zone of condensed counterions. As shown in Fig. 7, cable-like conductance through F-actin is characterized by the decomposition of an electric input pulse into delayed charge portions (solitons) indicative of discrete charge centers with corresponding counterion clouds along the polymer axis.

FIG. 7. Experiment of Lin & Cantiello (1993) to demonstrate cable-like conduction along actin "laments of 50 lmlength in aqueous 100 mM KCl. The diagram illustrates the output signal resulting from a 500 ls lasting square input pulse with an amplitude of about 2 na.

Formation of di!erent delayed discharge events can be explained by the capacitor-like action of the counterion clouds. At higher activation energies, more cations can accumulate within the clouds than necessary for neutralization of the corresponding "xed charge center. Consequently, ionic conduction systems of this type are equivalent to electron-conducting systems of coupled oscillators consisting of a resistance and a capacitor each. Both systems, the electron- and the ion-conducting system, exhibit nonlinear transmission characterized by delayed oscillating output signals. One of the main di!erences is the much higher time constants of ionic oscillations resulting from the slower migration velocity of ions in aqueous solutions compared with that of

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electrons in metallic conductors. Consequently, the output response in an F-actin bundle of 50 lm length is delayed by several seconds (Fig. 7). 3.5. THEORETICAL CONSEQUENCES

As discussed by Lin & Cantiello (1993), the nonlinear electrodynamics of F-actin may have far-reaching physiological consequences: &&One can envision an electric "eld #uctuation, such as elicited by a single channel opening to which an actin "lament is attached, as a novel and e$cient long-range intracellular signaling mechanism.'' If such considerations are valid for single cytoplasmic micro"laments, they are even more relevant for the membrane-shielded micro"lament bundles of microvilli, much more resembling isolated electric cables than free-#oating actin "laments. Since microvillar actin "laments are tightly associated with cation channels in the tip membranes, the microvillar conduction pathway may be essentially governed by the speci"c transduction properties of both ion channels and Factin. Thus, various well-known nonlinear channel characteristics may be produced by the microvillar cytoskeleton rather than by properties of the channel protein itself. Some of the theoretical implications following from the speci"c transduction properties of ions through microvillar actin "lament bundles are discussed in the following sections.

whereas movement of divalent cations between the "xed charge centers along the polymer axis a!ords much higher activation energy than transfer of K> or Na>. The same aspect of ionic selectivity is illustrated by another experimental approach. As shown by Tang & Janmey (1996), high ionic strength and, most e!ectively, di- and polyvalent cations reduce the repulsive action of the "xed charge on the polymer allowing side-by-side aggregation of actin "lament.s Association of actin "laments or bundles and bidimensional paracrystalline arrays was observed when the concentration of the divalent cations Ca> and Mg> was raised to 10 mM. The bundling e$ciency of divalent cations increases with their atomic number. Co> and Mn> is e!ective at 5 and 7 mM, respectively, and organic polycationic compounds are the most e!ective agents exhibiting bundling concentrations of 300 lM (Tang & Janmey, 1996). In contrast, addition of polyanions disaggregates bundles of single "laments. Hence, in the presence of divalent cations, loosely associated actin "laments aggregate into the more tightly aligned form in which the "xed charge centers are largely neutralized by polyvalent cations and ion conductance is signi"cantly reduced. Thus, currents through micro"lament bundles, preferentially carried by monovalent ions, can be largely modulated by the presence of the divalent cations.

3.5.1. In-uence of the charge number on ion conduction in actin ,lament bundles

3.5.2. Gating and recti,cation properties of the microvillar actin ,lament bundle

The selectivity of ion conduction through an ion exchanger matrix is generally given by the relative binding a$nity of the respective ion to the charged groups of the matrix. According to Carlier et al. (1986), the ATP-actin monomer binds 6}8 cations to low-a$nity binding sites. These sites exhibit KD values of 0.15 mM for Ca> and Mg> but 10 mM for K>. In actin polymerized under physiological conditions, these low-a$nity sites are likely to be occupied by Mg> and K> alone (Carlies et al., 1986). However, because of the two orders of magnitude lower binding a$nity of K> ions to the "xed charge centers of the polyelectrolyte charge transduction is preferentially carried by K> ions,

In a recent publication, Carbone et al. (1997) studied the Ca> and Na> permeability of neuronal channels permeable for Ca> and Na> (high-voltage activated L- and N-type). It is known that Na> and Ca> currents through these channels were blocked by micromolar and millimolar Mg>, respectively. On the other hand, Ca> ions possess the puzzling ability to block Na> currents through these Ca> channels at micromolar external concentrations but to permeate the channel when [Ca>]ex is raised to millimolar concentrations. In both cases, the degree of blockade increases with increasing negative potentials. The authors suggest that a divalent cation-binding site inside the channel

MICROVILLAR ION CHANNELS

is oriented in such a manner that the blocking Mg> (or Ca>) ion can enter the channel from either side, but is unable to permeate the channel completely. Reactivation of the channel occurs when the blocking cation is driven out of the channel pore by an adequate change of the membrane potential (Carbone et al., 1997). Similar voltage-dependent Mg> blocks from either side of the channels have been reported for Na> channels (Pusch, 1990; Pusch et al., 1989) and for Ltype Ca> channels of PC12 cells (Kuo & Hess, 1993). These gating properties of ion channels are in complete accordance with the microvillar di!usion barrier idea. Although the authors of these studies have proposed a channel model in which the inhibitory binding site is located within the channel pore, the data are also compatible with the properties of the cytoskeleton/channel complex existing in microvilli. Depending on the membrane potential, intra- or extracellular Mg> can occupy cation binding sites on either side of the actin "lament bundle, thereby forming a small high-resistance cap at either end of the "lament. Strongly negative potentials draw external Mg> (via non-selective cation channels) into the tip region of the microvillar "lament bundle, whereas strongly positive potentials force intracellular Mg> into the cytoplasmic end of the microvillar bundles (Fig. 8). Between these two threshold potentials, the channel is open for charge transduction by monovalent cations as indicated by the typical bell-shaped current/ voltage diagram. Thus, the microvillar F-actin matrix may form a structure with transistor-like properties, able to respond to changing electrical "elds with closure or opening of the ionic in#ux or e%ux pathway. As a consequence, following from the in#uence of the charge number on the conductivity in polyelectrolytes, "eld-induced di!usion of divalent cations into the microvillar F-actin bundle (gating current) is accompanied by an increased resistance for charge currents carried by the monovalent cations K> and Na>. For instance, the outward #ow of K> through the microvillar pathway is considerably inhibited by Mg> bound to the cytoplasmic end pieces of microvillar F-actin bundles. Increased invasion of cytoplasmic Mg> into the bundle structure

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FIG. 8. Potential-dependent Mg> block in the microvillar cation channel/cytoskeleton system. Positive or negative membrane potentials, cause electrodi!usion of Mg> ions into either side of the micro"lament matrix. Mg> bound to the low-a$nity sites of F-actin form blocking regions with high resistance for monovalent cation transduction.

can be expected under depolarizing conditions. This inwardly rectifying e!ect of Mg> and other polycations is a well-known property of certain K> channels, e.g. the ATP-dependent K> channel. Similarly, a cation in#ux-blocking (outwardly rectifying) e!ect would arise from external Mg> entering the microvillar tip region via nonselective channels of the tip membrane under conditions of hyperpolarization. The result of such &&gating'' e!ects of polyvalent cations is modulation of channel activity according to the actual electrochemical potential by formation of divalent cation caps at either the cytoplasmic or tip region of the conducting microvillar "lament bundle. Under normal physiological conditions (high cytoplasmic Mg>), Mg> caps the cytoplasmic end of microvillar micro"lament bundles. This Mg> cap largely prevents outward di!usion of K> via the microvillar pathway. It has recently been proposed that organic diamines such as spermin and spermidine can substitute Mg> as rectifying cation (Lopatin et al., 1994, 1995). Thus, in cells with microvillar surfaces cytoplasmic Mg> or polyamines (and Ca>) may act as e%ux blockers not only by occluding the channel pore but also, and perhaps most importantly, by inhibiting transmicrovillar cation conduction. Considering this &&transistor-like'' function of the microvillar actin "lament bundle, the physiological signi"cance of microvilli formation on the cell surface may be to protect the di!erentiated cell against excessive in#ux of either

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K. LANGE

physiological or toxic ions and to reduce the energy demand necessary for the permanent extrusion of these ions. On the other hand, reduced K> conductance critically lowers the membrane potential and o!ers the chance to use this potential in signal generation. 3.5.3. Membrane potential of cells with microvillar surfaces As a consequence of the resistance of "lament bundles to cation conduction, cell types with microvillar surfaces display relatively low (positive) membrane potentials, i.e. the K> di!usion potential is partly dissipated along the microvillar core structure (Fig. 4). Compelling evidence supporting this view comes from the observation that cells with microvillar surfaces (e.g. epithelial cells and hepatocytes) exhibit much more positive membrane potentials than the estimated K> potential of about !70 mV, which is consistently observed in cells lacking microvilli such as neurons, tumor cells, erythrocytes. The hepatocyte membrane potential, for instance, is reported to be around 30 mV (Wang & Wondergem, 1993a, b, 1991; Motzkin et al., 1990) and the potential di!erence across the syncytiotrophoblast microvillous membrane about 22 mV (Greenwood et al., 1993; Yamamoto, 1994; Birdsey et al., 1999). Similar low membrane potentials are observed in pancreatic b-cells (Wagooner et al., 1993) and endothelial cells (Vaca et al., 1996). In syncytiotrophoblast cells from human term placenta (Greenwood et al., 1993, Yamamoto et al., 1991), inwardly rectifying K> currents have been demonstrated and suggested that the low membrane permeability to K> and the low microvillous membrane potential di!erence may be a consequence of this type of inwardly rectifying channel (Greenwood et al., 1993). The role of microvilli in potential modulation is further supported by the "nding that receptormediated or thapsigargin-induced opening of the microvillar conduction pathway, as shown on the morphological level (Lange & Brandt, 1993a; Lange et al., 1997; Gartzke et al., 1997) is accompanied by hyperpolarization of the cell (Cabado et al., 1994; Vaca et al., 1996; Worley et al., 1994; Tarasiuk et al., 1995; Reiser et al., 1990; Bond

& Gordon, 1993). Hyperpolarization was also observed during hypo-osmotic swelling of hepatocytes (Wang & Wondergem, 1993a, b, 1991), which is accompanied by shortening of microvilli and integration of microvillar membrane portions into the enlarged cell surface (Sukhorukov et al., 1993; Cornet et al., 1994) [see Figs 10(a)}c)]. Wang & Wondergem (1993a) concluded, &&Hepatocyte transmembrane potential during osmotic stress responds as an osmometer in part because of changes in K> conductance.'' In accordance with the discussed inhibitory action of divalent cations on the microvillar cationic conduction pathways, cells depolarize in response to exposure to high external Mg> (Ibrahim et al., 1995). The pancreatic b-cell represents an excellent example to illustrate the role of the microvillar cation conduction system in signal generation. b-cells depolarize in the presence of high external glucose concentrations. Microvilli of these cells are equipped with a special type of low-a$nity glucose transporters (K "17 mM) that are exB pressed in di!erentiated cells only (Orci et al., 1989). As shown for di!erent cell types, high external glucose concentrations cause a conspicuous elongation of basal microvilli (Lange et al., 1990; Shepard et al., 1993, 1997; Shepard & Park, 1994), most likely due to cytoskeletal reorganization in response to increased ATP production. As a consequence of microvilli elongation on b-cells, the membrane potential becomes more positive giving rise to activation of voltagesensitive Ca> channels and subsequent insulin secretion. The special equipment of b-cells with low-af"nity glucose transporters (GluT2) acting at elevated glucose concentrations ('5 mM), as well as the localization of these transporters on microvilli are well established and represent the biochemical basis for the specialized function of this cell type in glucose homeostasis. The precise mechanism of glucose-induced microvilli elongation is still unclear. The exclusive location of the GluT2 transporters on microvilli points to an involvement of glycolytic ATP within the tip compartment. One of the best-documented examples of microvilli elongation in response to enhanced glucose supply has been reported by Shephard et al., (1993, 1997) and Shepard & Park (1994) for epithelial cells of the embryonic neural

MICROVILLAR ION CHANNELS

tube model. Thus, microvilli of pancreatic b-cells represent glucose sensors able to detect critical changes in external glucose concentration and to translate these changes into variations of the membrane potential. A considerable number of further adaptations of the microvilli to various physiological requirements are known. 3.5.4. Ion exchange properties of the microvillar pathway Transduction of ions through the microvillar pathway strongly depends on the type of ion channels present in the tip membrane. Generally, the polyelectrolyte nature of the microvillar shaft structure allows two di!erent types of ion #uxes: E

E

Microvilli containing cation channel but lacking anion channels are able to exchange cations between the cytoplasmic and external spaces. Several types of ion exchange processes are known such as Na>/H> and Ca>/Na> exchange, but evidence is also presented for exchange between the couples Ca>/H> and Mg>/Na>. However, Ca> and Mg> conduction is not likely to occur in the unstimulated microvillus but may proceed after receptor- or stress-mediated reorganization of the microvillar actin "lament into less dense structures such as orthogonal networks. The presence of anion and cation channels in microvilli tips allows co-transport such as NaCl transport (in epithelial cells) or cation/phosphate co-transport in addition to exchange processes. However, since the passage of anions through the negatively charged matrix is largely restricted, cation/anion cotransport is also con"ned to more permissive "lament arrangements.

As a consequence of the cytoskeletal modulation of microvillar ion transduction, receptor-mediated activation of ion #uxes via reorganization of the di!usion barrier characteristically alters the mode of ion transduction through this structure from the exchanger type to the co-transport mode. As shown by Lin & Cantiello (1993), the micro"lament system of microvilli is able to transduce monovalent cations in either direction according

571

to their electrochemical gradient. However, the barrier system exhibits considerable resistance against transduction of divalent cations and anions unless the bundle structure is modulated, either by depolymerization (shortening) or by reorganization into more permeable structures. Several types of cytoskeletal modulation have been demonstrated in response to receptor activation and other speci"c physiological signals. 3.5.5. Role of the structural organization of F-actin on channel activity 3.5.5.1. A¹P-dependent submembrane organization of the actin cytoskeleton. Regulation of glucose transport in adipocytes, glial cells, and hepatocytes is accompanied by changes in the length of microvilli and by transformation of microvilli into more voluminous and extended membrane structures such as ru%es and veils (Lange et al., 1989, 1990, 1998). Reorganization of the original, densely aligned "lament structure into less ordered arrangements signi"cantly impairs its function as di!usion barrier, resulting in stimulation of glucose/ion transport. Electron microscopic studies on C-6 glioma cells have shown that inhibition of glycolytic ATP production by iodoacetate or 2-deoxyglucose initially transforms microvilli into surface ru%es or veils allowing enhanced glucose uptake via microvillar transporters (Lange et al., 1989). Ru%es and veils were further shown to depend on the supply of ATP either of mitochondrial or glycolytic origin used for the formation of their internal networks of actin "laments. Scanning electron microscopy revealed that periods of high glucose uptake rates were accompanied by extensive veil ballooning, whereas in periods with low or almost blocked glucose uptake, veils collapsed onto the cell surface (Lange et al., 1989) (Fig. 9). Similarly, insulin stimulation of glucose transport was accompanied by ballooning of microvilli on the surface of adipocytes (3T3-L1) (Lange et al., 1990) and hepatocytes (Lange et al., 1998). Similar morphological e!ects can be produced on the surface of hepatocytes or HIT cells using agonist of the Ca> signaling system (vasopressin or bombesin) or thapsigargin known to activate Ca> in#ux into the cell (Lange & Brandt, 1993a;

572

K. LANGE

Lange et al., 1997). These "ndings strongly indicate that the uptake of ions or substrates via microvillar transporters/ion channels critically depends on the structural organization of the microvillar/submembrane cytoskeleton. As it appears, the di!usion barrier function of the dense bundle structure within normal microvilli is attenuated in F-actin networks allowing higher rates of in#ux into the cytoplasm. Reversible closure of these in#ux pathways also occurs by collapse of membrane foldings due to local ATP depletion. Restoration of local ATP concentrations by di!usion of cytoplasmic ATP reorganizes the cytoskeletal networks and opens in#ux pathways again. As shown for C6 glioma cells, this cyclic process gives rise to periodic changes in glucose uptake rates according to the scheme of Fig. 9. 3.5.5.2. Channel run-down and A¹P-dependent reactivation. Similarly, this novel principle of uptake regulation by cytoskeletal elements is able to explain the various "ndings of ion channel modulation by actin and actin-binding or severing proteins and chemical micro"lament e!ectors. Accroding to Fig. 9, the well-known e!ect of channel rundown in excised patches may be caused by depolymerization of micro"laments due to loss of actin monomers and ATP into the bathing medium, followed by collapse of the channel-containing membrane region. The reverse process of channel reactivation or inhibition of channel run-down by addition of ATP, actin monomers or short actin "laments has been observed for epithelial Na> channels (Berdiev et al., 1996; Maximov et al., 1997; Prat et al., 1993, 1996; Cantiello et al., 1991), ATP-dependent K> channels (Furukawa et al., 1996; Terzic & Kurachi, 1996), voltage-dependent K> channels (Wang et al., 1994; Jing et al., 1997), and Cl\ channels (Fatherazi et al., 1994; Prat et al., 1995). 3.5.5.3. Activation of microvillar channels by short actin ,laments2microvilli represent a barbed endshielded F-actin system. A series of patch clamp experiments have yielded evidence for an activating e!ect of short actin "laments on epithelial Na> channels (inside-out patches) (Berdiev et al., 1996; Maximov et al., 1997; Prat et al., 1993, 1996; Cantiello et al., 1991). On the basis of the

proposed concept, this "nding can be explained by two di!erent mechanisms depending on the state of the excised patch. Generally, the intact microvillar cytoskeleton can be characterized as a barbed end-shielded F-actin system. Microvillar actin "laments only expose their slowly growing pointed ends to the cytoplasm, whereas the rapidly growing barbed ends, located within the microvillar tip, are shielded from the cytoplasmic compartment. Because the barbed and the pointed ends display highly di!erent steady-state monomer concentrations, "laments exposing only their pointed ends cannot stably exist in the presence of "laments with free barbed ends. Therefore, barbed-end blockade of cytoplasmic F-actin by capping proteins is an inevitable precondition for the stable existence of microvillar surfaces. Under these conditions, the cytoplasmic concentration of free actin monomers is much higher than in the presence of barbed "lament ends. The generation or addition of "laments with free barbed ends immediately reduces the monomer concentration and initiates monomer dissociation from the pointed ends of microvillar "laments. This process results in rapid shortening or total disappearance of microvilli, accompanied by an activation of the respective uptake processes for ions or substrates. Addition of a high concentration of free barbed "lament ends to the cytoplasmic side of the patch (using short "laments) reduces by 1}2 orders of magnitude the concentration of free actin monomers in the immediate vicinity of the pointed "lament ends of microvilli (Fig. 10). Due to their high molecular weight, di!usion of actin monomers is a very slow process. Accordingly, depolymerization of F-actin in diluted or monomer-free solutions (as prevailing under patchclamp conditions) is always a slow &&di!usionlimited'' process. High concentrations of rapidly growing barbed "lament ends in the immediate vicinity of the pointed ends of the microvillar "laments considerably reduce the monomer concentration at these sites and, thereby, strongly accelerate monomer dissociation (Fig. 10). Insulin-induced activation of glucose and ion transport in hepatocytes (Lange et al., 1998) via activation of phosphatidylinositol (P1) 3-kinase (Lange et al., 1998) is an excellent example for this type of receptor-mediated signaling to the

MICROVILLAR ION CHANNELS

FIG. 9. ATP-dependent changes of the microvillar cytoskeleton accompany changes of the surface morphology. Low levels of intracellular ATP cause depolymerization of the microvillar cytoskeleton resulting in collapsed membrane folds. Subsequent ATP repletion initiates the growth of micro"lament networks blowing up the collapsed folds to voluminous blebs.

monomer depletion. Supply of actin (either as monomers or as F-actin) can reactivate microvillar channels by formation of submembrane actin networks unfolding channel-containing membrane areas (Fig. 9) as observed during transport oscillations in C-6 glioma cells (Lange et al., 1989). Numerous examples for modulation of ion channel activities by actin itself or by drugs modulating the structure of the actin cytoskeleton have recently been reviewed by Janmey (1998). Nearly all types of ion channels and exchangers can be a!ected by the actin-based cytoskeleton. Epithelial Na> channels, K(ATP) channel, voltage-dependent Na> channels, epithelial K> channels, cardiac K> channels, Cl\ channels (CFTR), Ca> channels, and Na>/H> exchanger were modulated by actin, cytochalasins or phalloidin. Thus, the microvillar di!usion-barrier concept of cell regulation adequately re#ects the present knowledge concerning the involvement of the actin cytoskeleton in ion channel regulation. In principle, the following types of cytoskeletal involvement are to be considered. E

FIG. 10. Activation of the microvillar pathway by addition of short F-actin "laments to the cytoplasmic side. The rapidly growing, barbed, ends of microvillar F-actin is shielded by the covering lipid membrane. In the presence of cytosolic barbed ends, the microvillar "laments depolymerize at their exposed slowly growing pointed ends, feeding growth at the barbed ends of added cytoplasmic F-actin.

microvillar e!ector system. Due to the massive formation of 3-phosphorylated phosphatidylinositols with very high a$nity to barbed endcapping proteins, a large number of barbed "lament ends were liberated within the cytoplasm which in turn shorten the microvillar "lament bundles. This rapid mechanism of micro"lament depolymerization is likely to be used in cellular signaling; it represents the physiological equivalent of the addition of short actin "laments to the cytoplasmic surface of excised membrane patches. Another situation is given when actin is added after channel run-down has occurred due to

573

E

E

Attenuation of ion #uxes by elongation of micro"lament bundles. Microvilli elongation is found in glucose-induced transport inhibition and may be involved in the inhibition of K>(ATP) channels in pancreatic b-cells. Microvilli elongation also represents a mechanism to modulate the membrane potential. Activation of ion #uxes by shortening of microvilli due to micro"lament depolymerization. This type of activation represents an initial event in receptor-mediated Ca> signaling (Lange & Brandt, 1993a; Lange et al., 1996, 1997), signaling via PI 3-kinase activation (Lange et al., 1998), and glucose starvationinduced transport stimulation (Lange et al., 1990). Activation of ion #uxes also occurs by reorganization of the bundle structure into networks of micro"laments "lling voluminous surface structures such as ballooned microvilli, ru%es or veils, as seen in response to receptormediated signaling (insulin and Ca> agonists) and during transport oscillation induced by inhibition of glycolytic ATP production in glial cells (Lange et al., 1989).

574 E

K. LANGE

Inactivation of ionic #uxes by collapse of ballooned membrane domains can be caused by breakdown of actin networks due to depletion of local [ATP] and/or actin monomers (Lange et al., 1989). This type of deactivation is observed under di!erent conditions of selective inhibition of glycolytic ATP production. Inactivation is readily reversed as soon as new ATP and/or actin is available; the newly growing "lament network again unfolds the collapsed membrane foldings (Lange et al., 1989).

E

3.5.6. Nonlinear charge conduction through microvilli The main result of the experiment of Lin and Cantiello (Fig. 7) is the demonstration of a characteristic type of nonlinear charge transduction through F-actin bundles. Charge transfer is not linearly related to the applied voltage and the current output at the end of the "lament bundle is discontinuous, i.e. during transduction, the input load is divided into several discharge pulses. Some of the electric properties of F-actin bundles closely resemble ion channel properties as visualized by electrophysiological techniques. E

Single-channel recordings always display discontinuous channel operation. Open periods are interrupted by periods of closed channels. The lifetimes of both states are variable within a wide range. Therefore, the conductance properties of ion channels have been described in terms of probabilities for the open and closed states. The participation of cytoskeletal structures in ion conductance may explain this type of channel operation in terms of discontinuous discharge properties of the involved micro"lament bundles. Especially, cation channels located at the tips of microvilli or membrane spikes may express the typical pattern of singlechannel recording Lin and Cantiello used actin "laments freely #oating in aqueous solutions allowing charge loss into the surrounding medium and, thereby, dampening of discharge oscillations. In the membrane-covered "laments of cell surface protrusions, this charge loss is prevented. Consequently, ion conduction through these structures should display a much sharper discharge contour, more close-

E

ly resembling the comb-like patterns of open and closed states in patch-clamp recordings. Ion transduction is limited by the capacity of ion channels at the microvillar tip. For instance, transduction in the outward direction may result in local accumulation of transduced ions within the tip compartment, causing transient closure of the whole conductance pathway due to chemical polarization within the microvillar tip compartment. The time period of this polarization-dependent closure of the whole pathway is largely determined by both the rate of ion transduction through the micro"lament bundle and that of ion e%ux via ion channels of the microvillar tip compartment. Using this picture, the open probability of the whole system not only depends on properties of the channel protein but also on conduction properties of the micro"lament bundle. The open probability would be lower when the conductivity of the bundle exceeds that of the microvillar channel(s). Gating currents, i.e. time-dependent deactivation of ion transduction, such as those observed in whole cell measurements of voltage-dependent Ca> and Na> channels may well be explained by rapid saturation of the binding sites of microvillar actin "laments with divalent cations (especially Mg>). Plugs of divalent cations at either side of the "lament bundle can inhibit further ion transduction through the structure as discussed in Section 5.2. In this way, divalent cations may be involved in recti"cation and voltage dependence of ion #uxes.

Summarizing the electric properties of organic hydrated polyanionic matrices, a general distinction can be made between those properties that are analogous to electronic semiconduction and those that are unique for &&ionic semiconductors''. Properties resembling semiconductors: E E

those

of

electronic

Linear polyelectrolytes exhibit high resistance under low-energy (basal) conditions. Applied external energy, e.g. thermal and (electric/magnetic) "eld energy increases the ion conductivity along the linear polyelectrolyte.

MICROVILLAR ION CHANNELS E

The activation energy for ionic #uxes is increased by dotation of the matrix with polyvalent cations. Field-induced installation of polyvalent ion plugs enables the F-actin bundle structure to function like an electronic switch or transistor.

Properties unique for &&ionic semiconductors'' of the F-actin type: E

E

E

E

Conductive resistance and activation energies are modulated by structural changes in the micro"lament system. Elongation, shortening, or reorganization of the bundle structure into "lament networks results in signi"cant changes of the conduction resistance especially for divalent cations. Due to the special dynamics of ion transfer between charge clouds (condensed ions) within the polyanionic matrix, oscillating transduction is possible. Such ionic conductance lines resemble electronic systems composed of a series of oscillators each consisting of a resistor and a capacitor. Because of the low migration velocity of ions in aqueous solution the time constants of ionic current oscillations are much larger than those of electronic oscillators. Reversible blocks can be established by capping the ends of the "lament bundle with polyvalent cations. Application of a small gating current, used for electrodi!usion of the &&cationic plug'' into the anionic matrix, modulates or shuts down conduction through the micro"lament bundle. The type of &&polyvalent plugs'' is determined by the selectivity of the cation channels in the microvillar tips and by the presence of divalent cations within the cytoplasm. As shown in the following section, the microvillar conduction system also represents an electro-mechanic and mechano-electric coupling device. 4. Mechanisms of mechanoelectrical coupling 4.1. MECHANO-ACTIVATION OF ION CHANNELS IN VOLUME REGULATION

Mechanosensitive or stretch-activated channels respond to tangential mechanical stress on

575

the membrane with an increase of conductivity. The most important type of membrane stress is generated by hypotonic cell swelling. In spite of extensive research, the mechanism of mechanoactivation of ion channels has remained obscure. In their latest review Sachs & Morris (1998) summarized the actual state of the problem in the following way: &&We are ignorant as to whether any eukaryotic MSC channel is mechanosensitive in an arti"cial lipid bilayer. There is increasing evidence that membrane structures beyond the channel are critical in signal conditioning of MSCs. Cytoarchitecture may render a channel either sensitive or insensitive to mechanical stimuli. Various aspects of mechanosensitivity depends strongly on the mechanical history of the channel-bearing membrane, as if cortical structures determine how mechanical energy is fed into the channels gating mechanism. But we do not known what cortical elements are involved.'' A recent study on several cell types has suggested that hypotonic cell swelling is always accompanied by a reduction in the number and length of microvilli. Membrane capacitance and cell rotation measurements were used by Sukhorukov et al. (1993) to study the e!ect of hypotonically induced changes in the plasma membrane of cultured mammalian cells and concluded: &&All measurements indicate that hypo-osmotically stressed cells obtain the membrane area necessary for swelling by using material from microvilli''. Similarly, PC12 cells respond within the "rst few minutes of hypo-osmotic shock with a decrease in the density and length of microvilli, which normally cover the cell surface (Cornet et al.,1994). These "ndings were con"rmed by morphological studies by the present authors on hepatocyte swelling. Scanning electron microscopy clearly showed a reduction of number and length of microvilli within 1 min of exposure to 200 mosm [Fig. 11(b)] and an almost complete recovery of the original surface morphology (RVD) within 10 min [Figs 11(c)]. Following cell swelling, regulatory volume decrease (RVD) is initiated by KCl e%ux through microvillar cation and anion (Cl\) pathways. The presence of these channels on microvilli has been demonstrated for hepatocytes (Lange et al. 1996), hamster insulinoma cells (HIT ) (Lange

576

K. LANGE

FIG. 11(a). Scanning electron micrograph of the surface of a freshly isolated rat hepatocyte cultured at normal osmolarity. (b) Scanning electron micrograph of a freshly isolated rat hepatocyte 1 min after addition of hypotonic medium (200 mosm). (c) Scanning electron micrograph of a freshly isolated rat hepatocyte 10 min after addition of hypotonic medium (200 mosm).

& Brandt, 1993b), as well as for the microvillous surface of placental syncytiotrophoblasts (Greenwood et al., 1993; Yamamoto, 1994; Birdsey et al., 1999; Yamamoto et al., 1991; Byrne et al., 1993; Dumaswala et al., 1993). Several further aspects of volume-activation can be readily explained on the basis of the microvillar channel concept. For instance, the low membrane potential, characteristic for the epithelial cell type with microvillicovered surfaces, is hyperpolarized on hypotonic, treatment. Swelling-induced hyperpolarization is not only observed in hepatocytes (Greenwood et al., 1993; Yamamoto, 1994) but also in placental syncytiotrophoblasts (Birdsey et al., 1999) indicating that the increase in cell volume is accompanied by an activation of ion current through K>(and Cl\ (Birdsey et al., 1999) channels on the microvillar cell surfaces. In some cell

types, cell swelling is accompanied by an increase in [Ca>] , most likely due to dilution-induced G Ca> release by monomer dissociation from Factin. Since enhanced [Ca>] itself, via Ca>G dependent actin-binding and -severing proteins, increases F-actin reorganization/depolymerization, the small dilution-dependent Ca> e!ect is potentiated, giving rise to increased ion e%ux via the microvillar pathways. On the other hand, release of cytoplasmic ATP has been shown to play a role in volume regulation via autocrine purinergic stimulation (Wang et al., 1996; Stroback et al., 1996; Beecroft et al., 1998; Rameh et al., 1998; Schlosser et al., 1996). ATP, leaving the cell via volume-activated anion channels, may potentiate the ion e%ux pathways to the extent required for volume recovery. Therefore, it is also possible that the observed

577

MICROVILLAR ION CHANNELS

volume-induced increase in [Ca>] is brought G about by the autocrine mechanism alone or at least potentiated by it. In some cases, mechanosensitivity is detected only when ion channels are pre-activated by receptor agonists. Thus, it appears that in some cell types, full channel activation cannot be achieved by volume-induced membrane stress alone and additional amplifying events, such as receptormediated Ca> signaling by released ATP is required or he complete activation of volumeregulating ion #uxes. The proposed microvillar mechanism of ATP release in volume regulation is supported by the detection of ATP-permeable e%ux pathways in microvilli of hepatocytes and HIT cells (Lange & Brandt, 1993b; Lange et al., 1996). According to this concept, the complete pathway of events involved in volume-induced channel stimulation and RVD may comprise the following steps (Fig. 12):

FIG. 12. RVD after hypo-osmotic cell swelling. Swellinginduced shortening of microvilli increases. ATP e%ux through anion channels. Purinergic activation potentiates opening of microvillar cation and anion channels to complete RVD.

(C) Regulation (R
Activated cation and anion channels release cytosolic K> and Cl\ as well as other volume relevant organic anions into the medium, driving water out of the cell until RVD is completed. The original microvillar cell surface is retored by regeneration of activated microvilli or formation of new microvilli from exocytic membrane domains [Fig. 11(c)].

(A) Initiation E

E

E

Uptake of water from the hypotonic medium into the cytosol generates a mechanical strain tangential to the cell surface su$cient to integrate part of the microvillar lipid membranes into the plasma membrane and to shorten the microvillar di!usion barriers. Cytosolic dilution results in depolymerization of F-actin and in liberation of F-actin-stored Ca>. Swelling-induced membrane stress results in microvilli shortening which partially activates microvillar pathways for Ca> in#ux and ATP e%ux. (B) Ampli,cation

E

E

Increased [Ca>] activates actin-binding and G severing proteins, which, by cytoskeletal reorganization, additionally contribute to the opening of microvillar ion pathways. ATP opens cation and anion in#ux pathways via purinergic (P2) receptor-coupled Ca> signaling (Wang et al., 1996; Strobaek et al., 1996) and inserts additional channel proteins into the plasma membrane via exocytosis.

In addition to volume regulation, mechanical channel activation plays an essential role in several other important physiological functions. For instance, blood pressure is regulated by the shear stress streaming blood exerts on the surface of vascular endothelial cells; bone formation is stimulated by osteocytes growing on mechanically charged bone structures; and the microvillar ion channels on audioreceptor cells (&&hair cell'') of the inner ear are activated by sonic waves. Clusters of microvilli on the surface of hair cells represent mechanoreceptors designed for converting mechanical energy into electrical signals. Since a large amount of structural and functional information about this specialized cell type has been accumulated, some relevant aspects of the mechanoreceptor system of hair cells will be discussed in the following section. 4.2. THE HAIR CELL MODEL OF MECHANO-ELECTRICAL COUPLING

The audioreceptor cell represents an extensively used experimental model for studying mechano-electrical coupling (Hudspeth, 1989; Ashmore, 1991; Sackin, 1995; Lumpkin & Hudspeth, 1995). This highly specialized cell type is

578

K. LANGE

FIG. 13. Acoustic stimulation of hair cell microvilli results in oscillating membrane potentials synchronized with sound frequency. Dislocation of the microvilli tips by only 10}50 nm signi"cantly increases the cation permeability of the hair cell microvilli. Thus, depending on the direction of dislocation the membrane potential changes by #10 of !10 mV (Huespeth, 1989). Mechano-electric coupling is reversible. Injection of currents into the cell generates microvilli dislocation (Crawford & Fettiplate, 1981).

equipped with a characteristic arrangement of large (apical) microvilli that can be laterally moved by applied acoustic "elds (Fig. 13). The swinging movement of this microvilli arrangement generates an oscillating membrane potential changing by about 10 mV around its normal value (E0) according to the direction of their dislocation. Using the fura-2 #uorescence technique, Lumpkin & Hudspeth (1995) recently demonstrated that even these highly specialized microvilli of hair cells have Ca>-permeable channels located at their tips. These channels were shown to be open in the resting state. Accordingly, enhanced Ca> concentrations were exclusively demonstrated in their tip compartments. Following sound-induced mechanical stimulation of the microvilli, a #ow of Ca> from their tip compartments toward their bases was observed. According to the increased cation conduction along the microvillar pathway, the hair cell membrane potential is changed. A possible mechanism of mechano-electrical coupling in microvillar F-actin bundles is shown in Fig. 14. Bending of microvilli gives rise to mutual sliding of the actin "lament within the bundle structure (Sachs & Morris, 1998). Filament gliding approximates the charge centers of neighboring "laments, thereby generating a molecular arrangement with higher energy content that facilitates the passage of a limited amount of counterions through the bundle. The general

FIG. 14. Mechanical induction of ion currents in F-actin bundles. Mutual dislocation of charge centers along the bundle axis facilitates the conduction of counterions along the whole bundle structure. Transmission occurs by charge transfer from one "lament to an adjacent one.

design of this mechanism is that of an ion switch operated by mechanical energy, a cellular device for coupling mechanical movement with the membrane potential. 4.3. REVERSE TRANSDUCTION IN HAIR CELLS (ELECTRO-MECHANICAL COUPLING)

Similar to other physical systems transforming mechanical into electrical energy, the mechanoelectrical coupling system of hair cells is able to work in the reverse direction (reviewed in Ashmore, 1991). Electro-mechanical coupling has been demonstrated using two di!erent experimental approaches. First, hair cells from di!erent locations on the auditory organ display a marked resonance of the potential di!erence generated in response to the (location-speci"c) sound frequency (potential resonance) (reviews in Hudspeth, 1989; Ashmore, 1991). Mechanical stimulation of hair cell microvilli with this selective frequency results in maximal amplitudes of the oscillating membrane potential. The same frequency of potential resonance is also generated by injection of a current pulse into hair cell (Tilney et al., 1983) (Fig. 15). Second, turtle hair cells respond to application of an electrical potential di!erence by injection of a small current pulse into the cell with lateral dislocations of microvilli in the nanometer scale (Crawford & Fettiplate, 1985). Since turtle hair cells show a pronounced potential resonance, the induced &&ringing'' in the stereocilial response suggests that such movements are potential driven. Similar observations were made with audioreceptor cells of eel, where transepithelial current leads

579

MICROVILLAR ION CHANNELS

FIG. 15. Tuned electrical resonance in hair cells (induced &&ringing''). Injection of a current pulse into the cell body provokes a damped oscillation of the membrane potential. The frequency of this oscillation is the same as that of sound to which the cell is most sensitive (Crawford & Fettiplate, 1981).

to a measurable de#ection of long stereocilia (Rusch & Thurm, 1989). These experiments clearly demonstrate that mechanical de#ections of the microvilli on hair cells are reversibly coupled with the respective electric oscillatory system. They also suggest that the electro-mechanical system of microvilli represents a cellular interaction site for external electromagnetic "elds at which "eld energy can be absorbed and transformed into mechanical or electrical responses.

5. Field-induced activation of microvillar cation channels 5.1. MAGNETIC FIELD INTERACTION WITH LINEAR POLYELECTROLYTES

Field interactions with cellular systems are not con"ned to electric "elds but are also possible with slowly changing magnetic "elds, which are able to induce a movement of condensed counterion clouds along the "lament axis. Displacement of the counterion clouds from their original position of lowest energy enhances the possibility of ion transfers from one "xed charge center of the "lament to the other and, thereby, facilitates cation conductance along the whole "lament. Torbet and Dickens (1984) demonstrated the alignment of actin "laments in a magnetic "eld, indicating that the condensed counterion system of actin "laments is able to convert magnetic "eld energy into mechanical movement. Since magnetic "elds can only interact with molecules exhi-

biting magnetic dipole moments, this "nding clearly indicates magnetic "eld-induced charge dislocation in actin "laments (induced magnetic moments). The e!ect closely resembles magnetic "eld-induced electron currents in semiconductors (Hall e!ect). The delineated mechanism of magnetic "eld coupling o!ers a possible systematic explanation of "eld-induced biological e!ects such as the most frequently observed impairment of Ca> signaling. 5.2. PHYSIOLOGICAL EFFECTS OF MAGNETIC FIELD EXPOSURE

Most epidemiological studies have concentrated on magnetic "eld exposure because, unlike electric "elds, magnetic "elds deeply penetrate the body tissue. Electrical "elds are much more e!ectively shielded by the dielectric properties of water-rich tissues than magnetic "elds. Recent studies have demonstrated a modest increase of risk for leukemia and brain cancer. Furthermore a list of suspected "eld-induced cancer types including male breast cancer and malignant melanoma has been put forward. Experiments with di!erent cell types consistently point to the Ca> signaling system as the primary cellular target of magnetic "eld interactions. The reported actions of magnetic "elds on Ca> signaling can be divided into two groups. In most studies, isolated cell systems were exposed to low-frequency (10}50 Hz) magnetic "elds. These experiments always yielded increased cytoplasmic Ca> levels (Barbier et al., 1996; Korzh-Sleptsova et al., 1995; Lindstrom et al., 1993, 1995; Fitzsimmons et al., 1994; Lidbury, 1992; Walleczek & Budinger, 1992; Lyle et al., 1991; Walleczek & Lidurdy, 1990; Carson et al., 1990; Papatheofanis, 1990). Some authors studied the e!ect of a static magnetic "eld on cell systems; in this case, Ca> in#ux was inhibited (Rosen, 1996; Zyost & Lidurby, 1992). On the other hand, a recent study has demonstrated that electromagnetic "elds severely a!ect the structural organization of microvilli. Pulsed electromagnetic "elds at frequencies between 50 and 70 Hz and a tension of 0.6 V cm\ resulted in the disappearance of microvilli and collapse of apical parts of endodermal cells in embryonic

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yolk sacks. The "eld-induced ultrastructural alterations were accompanied by severe embryotoxicity (Zusman et al., 1990). The same observation was reported by Santoro et al. (1997) on applying low-frequency magnetic "elds (50 Hz, 2 mT ) to human lymphoid cells. Field exposure resulted in a reorganization of the cortical actin cytoskeleton accompanied by loss of microvilli. These "ndings are consistent with the proposed magnetic "e ld interaction with linear polyelectrolytes of the microvillar cytoskeleton. Static magnetic "elds do not interact with resting electric charges but stabilize their spatial arrangement. Consequently, counterion clouds along the polyelectrolyte are spatially "xed and cellular Ca> uptake via the microvillar cation pathways is inhibited (Rosen, 1996; Yost & Lidurby, 1992). In contrast, changing electric or magnetic "elds may dislocate counterion clouds along the micro"laments, facilitating ion #uxes through the microvillar pathway. Due to the slow movement of ionic currents in aqueous systems, the microvillar ion conduction pathway is most e!ectively in#uenced by low-frequency magnetic "elds. According to the mechanism of electromechanical coupling shown in Fig. 13, charge dislocation along the "lament axis generates mutually repulsive charged centers in neighboring "laments aligned in the bundle structure. As shown in the reverse transduction experiment, microvilli respond to this induced state of intrinsic strain with an evasive deformation of the "lament bundle. The energy used for this deformation process is taken from the applied electric or magnetic "elds. Relaxation from this state of higher energy can be achieved in two di!erent ways. First, the produced mechanical stress may damage the subtle structural organization of basal (non-stabilized) microvilli, activating the cellular Ca> signaling pathway by impairment of the di!usion barrier function. Second, the intrinsic strain of the system can be relieved by ion translocation, generating a short current pulse through the "lament bundle similar to that observed during electro-mechanical coupling. Both ion #uxes as well as mechanical damage should be considered in further studies of "eld-induced biological hazards.

6. Conclusions The proposed hypothesis of ion channel function and regulation combines two main theoretical components: First, the concept of microvillar Ca> signaling (reviewed in Lange, 1999) depending on the following experimental "ndings: E

E

E

Segregation of transporter and channel proteins (and other di!erentiation-speci"c functional proteins) into the microvilli tips of di!erentiated cells. Function of microvillar actin "lament bundles as di!usion barrier for ions and metabolic substrates. Identi"cation of F-actin as IP3-sensitive and receptor-operated Ca> store and Ca> in#ux regulator.

Second, Manning's theory of &&condensed counterions'' in polymers of high charge density and Lin and Cantiello's experimental demonstration, that this theory is valid for F-actin under physiological conditions. The most important feature of the microvillar conduction system is its responsiveness to various receptor-mediated signals. Moreover, this conduction system exhibits unique functional #exibility which enables it to respond to various chemical and physical signals including electric and magnetic "elds. The localization of these ion-conducting organelles on the cell surface renders the ion pathways sensitive to a wide variety of mechanical stimuli. Specialized mechanosensor cells detect turbulent streaming on the surface of vascular endothelial cells; acoustic wave-induced #uid movement at the surface of the audioreceptor cells is transformed into changes of membrane potential; distortion of bone structure activates calcium phosphate deposition from osteoblasts; changes of cell volume due to altered environmental osmotic pressure activate ion channels for regulatory volume decrease. Other physiological adaptations of the microvillar system, e.g. photoreceptor and taste sensor cells, may function in a similar way. The functional relevance of microvilli has been severely underestimated in the past. One essential reason for this failure is the unexpected behavior

MICROVILLAR ION CHANNELS

of these cell organelles during homogenization by shear techniques, e.g. with Te#on}glass homogenators. Microvilli can be easily sheared o! from the cell surface, forming small membranebounded vesicles, which are indistinguishable by sedimentation techniques from membrane vesicles of intracellular origin such as endoplasmic reticulum or the Golgi system. Thus, a considerable number of biochemical systems that have been isolated in the microsomal fraction, are attributed to intracellular membrane systems although they are actually located on the cell surface. Up to now, only two of these &µsomal'' systems have been studied using the low-force shearing technique which is able to shear o! microvilli from the surface of the intact cell: the insulin-sensitive glucose transporter isoform GluT4 (Lange & Brandt, 1990a, b), and the IP3sensitive Ca> store (Lange & Brandt, 1993a, b). Originally, both systems were thought to be exclusively located in the endoplasmic reticulum or trans-Golgi vesicles. Although all membrane proteins primarily are components of the endoplasmic reticulum and the Golgi system because they are synthesized and processed there, the following exocytic insertion of trans-Golgi vesicles into the plasma membrane give rise to the hitherto unrecognized bifurcation of their further fate: in growing cells these proteins were evenly distributed over the whole cell surface, but in di!erentiated or resting cells, they become segregated into the tip membrane of microvilli (Lange & Brandt, 1990b). It is this novel aspect of cellular di!erentiation that still remains unrecognized although it is of eminent importance for cellular physiology. Most likely, several further microsomal systems may turn out to be functional components of microvilli in di!erentiated cells. Much further work will be necessary to assess the localization of the functional parts of these systems. REFERENCES ASHMORE, J. F. (1991). The electrophysiology of hair cells. Annu. Rev. Physiol. 53, 465}476. BARBIER, E., DUFY, B. & VEYRET, B. (1996). Stimulation of Ca> in#ux in rat pituitary cells under exposure to a 50 Hz magnetic "eld. Bioelectromagnetics 17, 303}311. BEECROFT, M. D. & TAYLOR, C. W. (1998). Luminal Ca> regulates passive Ca> e%ux from the intracellular stores of hepatocytes. Biochem. J. 334, 431}435.

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