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Ion Channels and Transporters on the Move

Albrecht Schwab

A. Schwab is at the Physiologisches Institut, Röntgenring 9, D-97070 Würzburg, Germany.

	Abstract

 
Cell migration plays a crucial role in a variety of (patho)physiological processes such as immune defense, wound healing, and formation of tumor metastases. Detailed models have been developed to describe cytoskeletal mechanisms of migration. However, evidence is accumulating that the activity of ion channels and transporters is also required for optimal cell locomotion.

	Introduction

Top Introduction Cytoskeletal mechanisms of... Ion channels and transporters... Ion channels and transporters... Perspectives References

 
Cell migration is an important physiological and pathophysiological process throughout life. From early in embryogenesis, cells are migrating through our bodies. Neural crest cells are migrating through the embryo to form the nervous system. Neuroblasts are moving within the central nervous system even after birth to reach their final place of work. Leukocytes are chasing invading bacteria or other pathogens, or they are crawling into sites of inflammation. Fibroblasts are migrating into a wound and thereby contributing to its closure. The same holds true for epithelial cells, which close smaller gaps in an epithelial layer by migrating along the basement membrane. Angiogenesis requires migration of endothelial cells, and atherosclerosis involves migration of smooth muscle cells. Finally, migrating tumor cells may be responsible for terminating life, since tumor cell migration is one of the crucial steps in the metastatic cascade.

The rates at which cells migrate vary considerably from cell type to cell type. Some epithelial cells or granular cells from the cerebellum move only a fraction of a micrometer per minute. The "professionals" such as neutrophil granulocytes are much faster: their rate of migration is 5–10 µm/min. Fish keratocytes are even faster than neutrophils. They can slide over their substratum at rates of up to 30 µm/min.

Despite these differences in function and speed, migrating cells share many similarities with respect to mechanisms of migration. A migrating cell is typically polarized within the plane of movement (Fig. 1). One can easily distinguish front and rear end, in particular when cells are crawling over a two-dimensional surface. The front is formed by a flat (~300 nm thin), organelle-free, fan-like process, the so-called lamellipodium. The rear end is formed by the prominent cell body that extends into a uropod. Different cytoskeletal mechanisms take place at the front and at the rear of migrating cells to allow efficient locomotion. Protrusion of the lamellipodium and retraction of the cell body do not always occur in parallel. The retraction of the rear edge may lag behind the extension of the lamellipodium in slowly migrating cells such as fibroblasts. Consequently, a cell can grow in length for several minutes before the cell body eventually "catches up." The clear separation between front and rear parts of migrating cells offers a great experimental advantage. The functional properties of these cell poles can be studied individually. For example, it is possible to direct inhibitors of membrane proteins to just one cell pole at a time (Fig. 2).

View larger version (110K): [in this window] [in a new window]   FIGURE 1. Video micrograph of a transformed renal epithelial Madin-Darby canine kidney (MDCK)-F cell that is migrating toward the upper right hand corner of the image. This image clearly shows the morphological polarization of a migrating cell. One can easily distinguish the lamellipodium (front) and the cell body (rear), which extends into a "tail." Width of the visual field, 100 µm.

View larger version (48K): [in this window] [in a new window]   FIGURE 2. The local inhibition of ion channels and transporters in lamellipodium and cell body elicits differential effects on cell migration. A: charybdotoxin (CTX), a blocker of Ca2+-sensitive K+ channels (IK1), inhibits migration only when it is directed toward the cell body (data taken from Ref. 13). B: in contrast, the inhibitor of the Na+/H+ exchanger EIPA impairs migration only when it is applied to the lamellipodium of migrating cells (modified from Ref. 4).

	Cytoskeletal mechanisms of migration

Top Introduction Cytoskeletal mechanisms of... Ion channels and transporters... Ion channels and transporters... Perspectives References

The cytoskeleton can only translocate a cell when the forces generated by the cellular "migration machinery" are transmitted to the surrounding extracellular matrix. When there is too little friction and cells can form no contacts with their substratum, locomotion is impaired. Similarly, locomotion is also impaired when the substratum is too "sticky" and cells cannot release their contacts. Thus the interaction between extracellular matrix and cell adhesion receptors has to be a highly coordinated process. It depends on the concentration of extracellular matrix proteins and the expression level of integrins, the cellular adhesion receptors (8). Moreover, the matrix contacts are dynamic and asymmetric. Thus a migrating cell forms new contacts at its front, and contacts are released at the rear of a cell. Sometimes, integrins are left behind (shed), so one can visualize a cell's path retrospectively by staining the shed integrins with the appropriate antibodies.

	Ion channels and transporters are involved in cell migration

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The function of ion channels and transporters is closely related to the actin cytoskeleton. On one hand, ion channels and transporters can be regulated by the state of actin filaments. This is of particular importance during cell volume regulation when swollen or shrunken cells try to restore their normal volume by means of activating ion channels and transporters. Volume regulation is impaired when the normal actin filament turnover is disturbed by drugs such as cytochalasin, which prevents the growth of actin filaments. In addition, the organization of the actin filament network by accessory proteins also modulates the ability of a cell to regulate its volume. Melanoma cells, which lack an actin filament cross-linking protein (ABP280), are unable to activate a K⁺ channel adequately during volume regulation (1). Thus the state of the actin cytoskeleton is of critical importance for the appropriate activation of ion channels and transporters during volume regulation.

On the other hand, changes of cell volume themselves in turn influence the actin cytoskeleton. Such volume changes can be elicited among others by activating or inhibiting ion channels and transporters. Cell swelling is accompanied by a disintegration of actin filaments, and cell shrinkage is followed by an assembly of actin filaments. The interdependence of actin filaments and cell volume indicates that the "correct" cell volume and thereby the "correct" activity of ion channels and transporters must play an important role in cell migration, a process critically relying on a rapid turnover of actin filaments. By setting the correct cell volume, ion channels and transporters create the intracellular milieu that is required for the optimal operation of the cytoskeletal migration machinery.

This concept has now been tested in several cell types for a number of ion channels and transporters. Human melanoma cells, which lack the actin cross-linking protein ABP280, are unable to migrate. Interestingly, they are also unable to activate K⁺ channels appropriately during volume regulation. Both defects are rescued by transfecting these cells with ABP280 (1). Conversely, migration of these rescued melanoma cells is impaired when Ca²⁺-sensitive K⁺ channels (IK1) are blocked. This type of K⁺ channel is required for migration of other cell types too (14). Thus IK1 channel activity appears to be a general requirement for cell migration. This conclusion is indirectly supported by the expression pattern of IK1 channels. They are predominantly found in cells with the ability to migrate. A different type of K⁺ channel, the voltage-dependent Kv3.1, was related to migration of embryonic nerve cells (3). K⁺ channels are, however, not the only ion channels found to be necessary for cell migration. Migration of granular cells from the cerebellum depends on the activity of N-type Ca²⁺ channels and N-methyl-D-aspartate (NMDA) receptors (5). Stretch-activated, Ca²⁺-permeable, nonselective cation channels are involved in migration of fish epithelial keratocytes (7). Locomotion of glioma cells (15) and of transformed renal epithelial [Madin-Darby canine kidney (MDCK)-F] cells is modulated by the activity of Cl^– channels.

Moreover, ion transporters also play an important role in cell migration. The Na⁺/H⁺ exchanger (NHE) is the best-studied transporter in this context. It is activated on chemotactic stimulation of neutrophil granulocytes, thereby facilitating their migratory response (10, 11). NHE activity is also required for migration of human melanoma and of MDCK-F cells, where it operates in parallel with the anion exchanger AE2 (4). The role of NHE for migration can at least partially be taken over by a H⁺-K⁺-ATPase in neutrophils and MDCK-F cells (4, 10). Finally, the Na⁺-K⁺-2Cl^– cotransporter has also been linked to migration of MDCK-F cells.

	Ion channels and transporters support migration in different ways

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Depending on their type, ion channels or transporters modulate the cytoskeletal migration machinery in one of two ways: by modifying either the [Ca²⁺]_i or the volume of migrating cells. Although these mechanisms will be discussed separately, they are probably closely interrelated with each other.

[Ca²⁺]_i. The [Ca²⁺]_i, which is higher in the rear part than in the front of migrating cells, is an important regulator for a number of cytoskeletal proteins. High [Ca²⁺]_i in the rear promotes the retraction of the rear part of a migrating cell. In contrast, low [Ca²⁺]_i in the front will favor the protrusion of the lamellipodium. This spatial component of Ca²⁺ signaling on the intracellular migration machinery has superimposed on it a temporal component involving fluctuations in [Ca²⁺]_i in migrating cells. Thus the alternating predominance of mechanisms leading to the retraction of the rear part or to the protrusion of the lamellipodium is necessary for optimal cell locomotion. Accordingly, migration of several cell types requires oscillations of [Ca²⁺]_i (5, 7). Migration of cerebellar granule cells depends linearly on amplitude and frequency of fluctuations of [Ca²⁺]_i. These fluctuations in turn can be modulated by the activity of N-type Ca²⁺ channels or NMDA receptors. Blocking these channels decreases the rate of migration (5). Migrating fish keratocytes also display transient increases of [Ca²⁺]_i. These Ca²⁺ elevations coincide with phases of increased membrane tension. Membrane tension rises because the rear edge of the cell is stuck to the substratum while the lamellipodium keeps on protruding and thereby stretches the cell membrane. The increase in membrane tension activates a Ca²⁺-permeable, stretch-activated cation channel. The resulting transient rise of [Ca²⁺]_i then triggers those processes leading to retraction of the rear edge of migrating keratocytes (7). The stretch-activated cation channel thereby indirectly controls the cytoskeleton of a migrating cell.

Cell volume.
Transient increases of [Ca²⁺]_i also activate IK1 channels in migrating cells. Studies of volume regulation show that activation of these K⁺ channels is followed by cell shrinkage. Such volume loss due to activation of IK1 channels also occurs in migrating cells. Oscillations of [Ca²⁺]_i trigger the intermittent loss of up to 20% of the cell volume (12). Since blockade of IK1 channels prevents volume fluctuations and inhibits migration, we postulated that K⁺ channel-mediated volume fluctuations are part of or modulate the cellular migration machinery. In a similar way, a Cl^– channel was suggested to modulate migration of glioma cells (15). The question that arises is how intermittent volume loss contributes to cell migration. A clue comes from experiments in which we applied the scorpion venom charybdotoxin, a blocker of IK1 channels, exclusively to the lamellipodium or to the cell body of migrating cells. Migration is only inhibited when the rear part of a crawling cell is exposed to the blocker (Ref. 13; Fig. 2A). Consequently, IK1 channel-mediated volume changes almost exclusively affect the rear part of migrating cells (12). Thus IK1 channels facilitate the retraction of the rear part of migrating cells by inducing a local cell shrinkage at this cell pole.

If local cell shrinkage is associated with the retraction of the rear part of migrating cells, it appears logical that the protrusion of the lamellipodium would be accompanied by a local cell swelling. In such a scenario, transporters mediating solute uptake and, thereby, osmotically obliged water influx had to be present in the lamellipodium. NHE1 and AE2 are two of the candidate transporters. Both transporters are required for optimal cell migration (4, 10, 11). Indeed, they are both concentrated at the leading edge of the lamellipodium of migrating cells (2, 4). NHE1 colocalizes with proteins of cell adhesion complexes at this cell pole (2). Immunocytochemical localization of the exchangers matches very well with functional data. As depicted in Fig. 2B, inhibitors of NHE1 or AE2 impair migration only when they are directed to the lamellipodium. They have no effect on migration when directed to the rear part of a migrating cell (Ref. 4; Fig. 2B).

On the basis of these findings, we propose the following cycle of events to explain the contribution of ion channels and transporters to cell migration (Fig. 3). Starting after a transient rise of [Ca²⁺]_i, cell volume and IK1 channel activity have reached a minimum. Low [Ca²⁺]_i and a shrunken cell volume could favor actin filament polymerization and the outgrowth of the lamellipodium. Cell volume is replenished by NHE1 and AE2 activity, among other processes. Since these transporters are active at the front of migrating cells, they act in concert with gelosmotic swelling at the leading edge of the lamellipodium. The extending lamellipodium and the gradual cell swelling increase the tension of the plasma membrane and eventually activate Ca²⁺-permeable, mechanosensitive cation channels. [Ca²⁺]_i rises, leading to the activation of IK1 channels. The resulting local shrinkage of the rear part facilitates Ca²⁺-sensitive cytoskeletal mechanisms underlying the retraction of this pole of a migrating cell. After volume loss and retraction of the rear part, mechanosensitive Ca²⁺ entry stops, [Ca²⁺]_i returns to basal levels, and the cycle starts all over again. In a simplified view, one can therefore describe migration as temporally and spatially separated phases of local cell swelling and cell shrinkage.

View larger version (29K): [in this window] [in a new window]   FIGURE 3. Model summarizing the function of ion channels and transporters in migrating cells. Salt and osmotically obliged water uptake mediated by the parallel operation of Na+/H+ and Cl–/HCO3– exchangers at the front of migrating cells contribute to the extension of the lamellipodium (A and B). Increasing volume and membrane tension eventually trigger a rise of intracellular Ca2+ concentration ([Ca2+]i) via activation of Ca2+-permeable, stretch-activated cation channels (C). The rise of [Ca2+]i induces the retraction of the rear part of a migrating cell, which is paralleled by massive K+ efflux and local cell shrinkage at the rear (C and D).

	Perspectives

Top Introduction Cytoskeletal mechanisms of... Ion channels and transporters... Ion channels and transporters... Perspectives References

	Acknowledgments

 
Work cited from this laboratory was supported by Deutsche Forschungsgemeinschaft SFB 176, A6, and Schw 407/7-1.

	References

Top Introduction Cytoskeletal mechanisms of... Ion channels and transporters... Ion channels and transporters... Perspectives References

 

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