Pflugers Arch - Eur J Physiol (2007) 453:421–432 DOI 10.1007/s00424-006-0138-6

INVITED REVIEW

Cells move when ions and water flow

Albrecht Schwab & Volodymyr Nechyporuk-Zloy & Anke Fabian & Christian Stock

Received: 30 June 2006 /Accepted: 9 July 2006 / Published online: 5 October 2006 # Springer-Verlag 2006

Abstract Cell migration is a process that plays an life cycle. Migration starts early on during embryogenesis. important role throughout the entire life span. It starts early After birth, cells like neuroblasts still need to move to their on during embryogenesis and contributes to shaping our final “place of work” [60]. The outgrowth of nerve growth body. Migrating cells are involved in maintaining the cones can be viewed as a special form of cell migration integrity of our body, for instance, by defending it against since the cell body of the nerve cell usually remains invading pathogens. On the other side, migration of tumor stationary [41]. Wound healing requires the movement of cells may have lethal consequences when tumors spread fibroblasts or epithelial cells. Epithelial wounds frequently metastatically. Thus, there is a strong interest in unraveling occur in the mucosa of the gastrointestinal tract, and the cellular mechanisms underlying cell migration. The migration of epithelial cells into the denuded area is a fast purpose of this review is to illustrate the functional way to reestablish epithelial integrity [20]. Other processes importance of ion and water channels as part of the cellular strongly dependent on cell migration are the responses of migration machinery. Ion and water flow is required for the immune system, angiogenesis [127], and the formation optimal migration, and the inhibition or genetic ablation of of tumor metastases [144]. Thus, migrating cells originate channels leads to a marked impairment of migration. We from a broad range of different tissues, and they fulfill briefly touch cytoskeletal mechanisms of migration as well diverse physiological and pathophysiological functions. as cell–matrix interactions. We then present some general Yet, the cellular mechanisms underlying migration of these principles by which channels can affect cell migration different cell types often follow common rules. Some of before we discuss each channel group separately. these common mechanisms will be briefly summarized before we discuss the function of activity for Keywords . Cell migration . Cell–matrix cell migration in more detail. interactions . Cytoskeleton . Ion channels

Migrating cells are polarized Introduction All migrating cells are polarized morphologically and Cell migration is required for embryogenesis [11] and the functionally along the axis of movement [75, 86]. This is well-being of our body throughout life. On the other hand, particularly evident when cells are migrating on a two- “too much” migration or migration of the wrong cell type dimensional substrate. The front part of migrating cells is may be the cause of serious health problems or death. There formed by the so-called lamellipodium that is a fan-like, is hardly any cell that does not migrate at a given time in its 300-nm thin, and organelle-free process [1]. The cell body : : : that may extend into a uropod marks the rear part. The A. Schwab (*) V. Nechyporuk-Zloy A. Fabian C. Stock ability to maintain this polarization is a prerequisite for Institut für Physiologie II, Universität Münster, directed migration. It implies that different components of Robert-Koch-Str. 27b, 48149 Münster, Germany the cellular migration machinery are active at either cell e-mail: [email protected] pole. A repeated and coordinated cycle of protrusion of the 422 Pflugers Arch - Eur J Physiol (2007) 453:421–432 lamellipodium and retraction of the rear part of the cell will matrix [9]. Focal contacts comprise heterodimeric integrins then result in directed migration. In chemotactically composed of α and β subunits [43], the focal adhesion stimulated cells such as Dictyostelium discoideum or kinase (FAK; [122]), talin, vinculin, paxillin, and other neutrophil granulocytes, the enzymes phosphatidylinositol- attached to the actin filament network [10, 147]. 3 kinase (PI3K) and phosphatase and tensin homologue in FAK becomes phosphorylated upon focal adhesion forma- 10 (PTEN) play a crucial role in maintaining tion when integrins bind to the extracellular matrix. It helps the polarity [80]. They lead to the localized accumulation of coordinate the signal transduction events activated down- phosphatidylinositol-3,4,5-triphosphate at the leading edge stream of both integrins and growth factor receptors [42]. of chemotaxing cells. That is, a shallow gradient of an The turnover of integrin-mediated adhesions, especially extracellular chemoattractant can be amplified into a steep the release of cell–matrix contacts at the rear end, is a intracellular gradient of phosphatidylinositol-3,4,5-triphos- limiting process during cell migration. Tyrosine-kinase- phate. This modulates actin remodeling at the leading edge mediated phosphorylation and proteolysis of focal adhesion and myosin II assembly at the rear part of motile cells. In components by the calpain family of intracellular proteases addition, the Na+/H+ exchanger NHE1 is also involved in contribute to the adhesion turnover [14]. Thus, calpain- maintaining the polarity of migrating cells. Accordingly, mediated cleavage of the focal adhesion component talin is NHE1-deficient cells have a severely impaired ability for involved in controlling the turnover of integrin-dependent persistent migration into one direction [18, 94, 121]. focal adhesions [28]. Another mechanism controlling cell adhesion depends on the extracellular pH and involves the activity of the Na+/H+ exchanger NHE1 [126]. As NHE1 and the integrins are colocalized at the leading edges of Cytoskeletal mechanisms of cell migration lamellipodia [19, 34, 54, 98], NHE1 creates a proton- enriched nanoenvironment in the immediate vicinity of the

The cytoskeleton is probably the most important cellular focal adhesion complexes. The local pHe at focal adhesion motor for cell migration. The lamellipodium comprises a sites modulates the strength of cell adhesion and thereby dense meshwork of actin filaments [35]. They are poly- migration on a collagen I matrix [126]. A surplus of protons merizing at their plus ends into the direction of movement or a high NHE1 activity leads to a tight adhesion and and thereby protrude the plasma membrane at the leading eventually, if strong enough, to a decrease in cell migration, edge of the lamellipodium [82, 137]. Actin monomers and whereas a lack of protons due to low NHE1 activity filaments are escorted by many proteins that ensure the prevents adhesion and migration (see [125] for a review on fine-tuning of actin filament remodeling according to the the function of NHE1 in cell migration). needs of the crawling cell [141]. The Arp2/3 complex catalyses for example the nucleation of new actin filaments and the growth of existing filaments [148]. It is activated by members of the WASP/WAVE family [107]. These proteins Modulation of cell migration by ion channels are regulated by diverse factors, such as the phospholipid phosphatidylinositol-4,5 bisphosphate (PIP2), calmodulin, We will first give a general overview of possible mecha- Ca2+, and by the family of small Rho GTPases, e.g., Cdc42 nisms by which ion channel activity can modulate the or Rac, that promotes the formation of specific cellular cellular migration machinery before discussing the role of morphologies and plays a major role in cell migration [96, individual ion channels in more detail. The close interrela- 99, 146]. The retraction of the rear part of a migrating cell tion between ion channel activity and cell migration is in is mediated among others by the contraction of the cortical part due to one of the housekeeping functions that ion actomyosin network at this cell pole so that inhibition of channels exert in almost every cell. Ion channels play a myosin II leads to reduced motility of neutrophils due to pivotal role in cell volume regulation [64]. This is an defective uropod detachment [24]. important issue for cell migration since the integrity of the actin cytoskeleton is critically dependent on cell volume. Cell swelling leads to actin depolymerization, whereas cell Cell–matrix interactions during migration shrinkage promotes actin polymerization [37, 95, 119]. Thus, ion channels are crucial for creating the “correct” Coordinated formation and release of focal adhesion intracellular milieu for the optimal functioning of the contacts to the extracellular matrix mediated by integrin cytoskeletal migration machinery. The interrelation between receptor molecules are needed for cell migration [48] (for ion channel activity and cell migration becomes even more review see [139]). Focal contacts mediate the force intricate by the fact that cytoskeletal components them- transduction from the cytoskeleton onto the extracellular selves regulate ion channel activity [23, 35, 77, 90] so that Pflugers Arch - Eur J Physiol (2007) 453:421–432 423 the ability of ion channels to regulate cell volume relies on the transepithelial potential is absent [104]. Similarly, tumor an intact cytoskeleton. Another intriguing aspect is that cell migration and the sprouting of endothelial cells are some ion channels expressed in migrating cells and the affected by electric fields [85, 140]. actin cytoskeleton are regulated by the same phospholipid, Finally, several studies show that ion channels directly PIP2 [69, 89, 109, 146]. However, the functional signifi- interact or are colocalized with important proteins of the cance of this parallel regulation for cell migration has yet to cellular migration machinery [5, 15, 67, 90]. A common be determined. theme of these studies is that ion channels modulate cell Cell volume of migrating cells is likely to be subject to adhesion. fluctuations that are related to permanent shape changes during migration. Thus, the protrusion of the lamellipodium and the retraction of the rear part do not always occur K+ channels simultaneously in migrating cells. Rather, one of these processes temporarily dominates so that the cell volume Different types of K+ channels have been linked to may rise during the protrusion of the lamellipodium [110, migration. A prominent role is played by Ca2+-sensitive + 125], whereas it may decrease during the retraction of the K channels (IK1 or KCa3.1; [44]). The importance of these rear part. However, in the long-term, volume regulatory channels for migration is underlined by the fact that they mechanisms involving ion channel activity assure that the are expressed in probably all migrating cells including all cell volume remains constant. leukocytes and many tumor cells [3, 25, 47, 55, 81, 92, 93, In addition to setting the cell volume, ion channels are 115, 129]. Their inhibition slows down migration, whereas critical for cell migration since they are key players in the their heterologous expression speeds up cells that do not 2+ 2+ regulation of the intracellular Ca concentration ([Ca ]i). contain endogenous IK1 channels [50, 113, 119, 122] 2+ 2+ Cell migration is a Ca -dependent process [56, 96]. For (Table 1). IK1 channels are activated by a rise of [Ca ]i 2+ 2+ example, Ca modulates the turnover of actin filaments. A [44, 142]. Since [Ca ]i is oscillating in many migrating 2+ simplified view is that an elevation of [Ca ]i promotes cells, the activity of IK1 channels fluctuates [116]. That the 2+ 2+ + actin depolymerization, whereas a low [Ca ]i favors actin activity of Ca -sensitive K channels is fluctuating is 2+ polymerization. The intracellular gradient of [Ca ]i with important for their impact on migration because cell the lower concentration at the cell front [12, 36, 118] motility is impaired when the activity of Ca2+-sensitive thereby contributes to localizing actin polymerization to the K+ channels is kept at a constant level by K+ channel lamellipodium. Other examples of Ca2+-dependent process- blockers or activators [61, 120]. The Ca2+ sensitivity of IK1 es relevant for cell migration are the involvement of channels provides a way to coordinate their activity with calpains in the release of cell–matrix contacts [27], other Ca2+-dependent mechanisms of migration outlined 2+ recycling of integrins [65], or phosphorylation of the above. Due to the intracellular gradient of [Ca ]i, IK1 myosin light chain [145], which all support the retraction channels are active only at the rear part of migrating cells. 2+ of the rear part. Indeed, transient increases of [Ca ]i occur This conclusion was drawn from experiments in which IK1 shortly before the retraction of the rear part of migrating channel blockers were applied topically either to the front cells [21]. Since the cell membrane potential has a strong or to the rear part of migrating cells [117]. Migration was impact on Ca2+ influx—by controlling channel gating and/ only inhibited when blockers were applied to the rear part or by determining its electrochemical driving force—the of migrating cells. Accordingly, activation of IK1 channels contribution of ion channels to setting the cell membrane results in a localized shrinkage of migrating cells at their potential represents another way by which they could trailing edge, thereby supporting the retraction of this cell indirectly affect cell migration. However, despite the pole [114]. In osteoclasts, IK1 channels are also involved in 2+ importance of [Ca ]i as a coordinator of different compo- cell spreading [26] (Fig. 1). nents of the migration machinery, relatively little is known Several K+ channels have been shown to be concentrated about the Ca2+ channels mediating Ca2+ influx. at the front of migrating cells [52, 105, 122]. The functional Ion channels were also proposed to act as antennas that significance of this characteristic distribution has not measure electrical fields and thereby affect galvanotaxis always been determined. Kv1.3 channels in neutrophil [52]. It is known for a long time that electric fields can granulocytes were proposed to act in concert with TRPC1 direct migration [78, 85, 91]. This is particularly important channels as sensors for extracellular electrical fields. for migration of epithelial cells during the wound healing TRPC1 channels are also concentrated at the leading edge process. Epithelia generate transepithelial potentials of up [52, 53]. In contrast, the concentration of IK1 channels at to several tens of millivolts so that epithelial migration the cell front has not yet been correlated with a specific during wound repair occurs in an electric field that extends function for cell migration. They appear to be inactive at from the intact epithelium to the injured epithelium where this cell pole [122]. 424 Pflugers Arch - Eur J Physiol (2007) 453:421–432

Table 1 Ion and water channels involved in cell migration

Channel Cell type Function in cell migration Reference

K+ channels IK1 MDCK-F cells Blockade slows down, local reduction in cell volume at the [116, 120, 122] rear part of migrating cells MDCK cells Blockade slows down [50] Fibroblasts Blockade slows down [119] Melanoma cells Blockade slows down [119] Microglia Blockade slows down, prevents chemokinetic stimulation [113] Osteoclasts Cell spreading [26] HEK293 Heterologous expression accelerates [122] BK Glioma Activation and inhibition slow down [61, 79] 2+ Kv1.1, Kv1.5 Intestinal epithelial cells Expression elevates [Ca ]i and accelerates wound closure [101] Kv1.3 Lymphocytes Cell adhesion, co-immunoprecipitates with integrins [67]

Melanoma cells Interacts with β1-integrins of adhering melanoma cells [5] Neutrophil granulocytes Detection of electric fields and their coupling to metabolic [52] oscillators Cl− channels VRAC Glioma cells Cell shrinkage enhances invasive capacity [100, 124] Monocytes Blockade inhibits chemotaxis [51] Neutrophil granulocytes Blockade inhibits transendothelial migration [83] ClC3 Glioma cells Internalization inhibits migration [79] Aquaporins AQP1 Endothelial cells Supports tumor angiogenesis, enhances lamellipodial [110] protrusive activity Proximal renal epithelial cells Accelerates epithelial restitution [38] AQP4 Astroglial cells Promotes glial scar formation, enhances lamellipodial [111] protrusive activity AQP9 Neutrophil granulocytes Enhances lamellipodial protrusive activity [72] Ca2+ channels Mechanosensitive Keratinocytes Activation induces retraction of the rear part [66] Fibroblasts Blockade at cell front inhibits migration and traction force [84] generation TRPC1 Intestinal epithelial cells Accelerates epithelial restitution [102] Neutrophil granulocytes Electric field detection [52] Xenopus spinal neurons Guidance of nerve growth cones [123, 136] TRPV4 Hepatoblastoma cells Accelerates migration [138] TRPV1 Hepatoblastoma cells Accelerates migration [138] Dendritic cells Stimulates migration to draining lymph nodes [6] TRPM2 Neutrophil granulocytes Chemotaxis [39, 40] TRPM7 HEK293 cells Cell adhesion via calpain [128] HEK293 cells, N1E-115 cells, PC12 cells Cell adhesion via actomyosin [15] L-type Fibroblasts Contraction of trailing tail [145] N-type Cerebellar granule cells CNS development [57] NMDA-receptor Cortical neurons CNS development [8] Cerebellar granule cells CNS development [58] Voltage-gated Glial cells CNS development [71] Na+ channels Voltage-gated T lymphocytes Invasion [30] Mammary carcinoma cells Promotes motility and metastases [31] Prostate cancer cells Enhances motility [29]

Nav1.2, 1.6, 1.7 Neoplastic mesothelial cells Promotes motility [32] ASIC1 Astrocytes, glioblastoma cells Enhances motility [134] Pflugers Arch - Eur J Physiol (2007) 453:421–432 425

Fig. 1 Schematic representation of mechanisms by which ion and polymerization. c Mechanosensitive Ca2+ influx (possibly via TRPC1 water channels influence cell migration. a The activation of Ca2+- or TRPV4 channels) is an important trigger for the retraction of the + − 2+ sensitive K channels (IK1) and Cl channels (possibly VRAC or rear part of migrating cells. The rise of [Ca ]i leads among others to ClC3) mediates the efflux of K+ and Cl− ions. The resulting localized the activation of calpain proteases and the subsequent disassembly of shrinkage of the rear part of the cell supports cytoskeletal mechanisms focal adhesion contacts. TRPM7 channels lead to the activation of underlying its retraction. IK1 channels are activated by a (transient) calpain proteases or regulate actomyosin contractility. Both processes rise of the intracellular Ca2+ concentration. b The protrusion of the affect cell adhesion. d Kv1.3 channels are concentrated at the leading leading edge of the lamellipodium critically depends on the directed edge of the lamellipodium. They are required for the activation of β1- polymerization of actin filaments. The uptake of Na+ and Cl− ions by integrins. Clustering of Kv1.3 channels at this cell pole plays an the Na+/H+ exchanger NHE1 and anion exchanger AE2 at the cell important role in the detection of extracellular electric fields and front creates an osmotic gradient triggering the local influx of water downstream effects relevant for cell migration. Voltage-gated Na+ via aquaporins (AQPs). The increased local hydrostatic pressure channels were also proposed to detect electric fields and thereby affect protrudes the cell membrane and thereby makes room for actin galvanotaxis

In lymphocytes and human melanoma cells, Kv1.3 leukemia cells to a fibronectin matrix [4]. Interestingly, the + channels are physically interacting with β1-integrins and functional relation between K channels and β1-integrins is thereby control cell adhesion [5, 67]. The physical reciprocal. Blocking K+ channels also inhibits integrin- interaction between K+ channels and integrins may be the mediated cell adhesion. These studies did not explicitly cause for K+ channel activation upon adhesion of erythro- address the significance of the K+ channel–integrin inter- 426 Pflugers Arch - Eur J Physiol (2007) 453:421–432 action for cell migration. However, given the importance of exchanger NHE1 and the anion exchanger AE2 are also a coordinated formation and release of cell–matrix contacts concentrated at the cell front [19, 34, 54, 63]. Consequently, for cell migration, such a role appears likely. the overexpression of AQP1 and AQP4 augments the The expression of voltage-gated K+ channels (Kv1.1 and protrusive activity of the lamellipodium, and their knockout Kv1.5) increases the rate of epithelial restitution in part due impairs tumor angiogenesis, epithelial restitution after an to their influence on the intracellular Ca2+ concentration. acute renal failure, and astroglial scar formation [111]. K+-channel-mediated hyperpolarization of the cell mem- AQP9 was found to be involved in migration of neutrophil brane potential increased the intracellular Ca2+ concentra- granulocytes. tion due to an elevated electrochemical driving force for According to the Brownian ratchet model, the bending Ca2+ influx [101]. of actin filaments due to thermal fluctuations makes room for actin monomers to polymerize onto their barbed ends. This allows the elongated actin filaments to recoil and exert Cl− channels an elastic pushing force on the membrane and thereby protrude the leading edge of the lamellipodium [82]. Solute There are only few studies investigating the role of Cl− and water influx at the leading edge would greatly support channels in cell migration. There is a clear necessity for Cl− the ratchet mechanism by providing a force that extends the channel activity in migrating cells since K+ channels can plasma membrane. This raises the important question of only affect cell volume when K+ ions are accompanied by how changes of cell volume can be restricted to the cell an anion [116]. If Cl− channels were to work in concert front. Possibly, the dense actin meshwork within the with K+ channels in order to induce volume changes during lamellipodium serves as a valve since it slows down cell migration, volume-regulated Cl− channels (VRAC) osmotic water flow [45]. Thus, the dissipation of a localized would therefore be good candidates. However, it has to be volume increase at the front is at least delayed by this noted that the exact molecular nature of VRAC is a matter mechanism. A theoretical analysis revealed that an osmotic of debate [49, 87]. Nonetheless, several studies showed the gradient can indeed elicit water fluxes that result in the involvement of VRAC in cell migration. Migration of movement of cells [46]. glioma cells depends on the activity of a Cl− channel that is activated by cell swelling [100, 124]. Inhibition or internalization [79] of these channels, possibly ClC3, slows Ca2+ channels down migration and invasion of glioma cells. A model was suggested according to which Cl−-channel-dependent cell Cell migration is a Ca2+-dependent process. There is a strict shrinkage facilitates glioma cell migration through the necessity for a tight regulation of the intracellular Ca2+ 2+ dense extracellular matrix. Recent studies with human concentration ([Ca ]i). Many migrating cells exhibit (−/−) 2+ monocytes or neutrophil granulocytes from Clcn3 mice oscillations of [Ca ]i [21, 73]. In addition, there are spatial 2+ 2+ came to a similar conclusion. Inhibition of the swelling- differences of [Ca ]i in migrating cells. [Ca ]i is higher at activated Cl− channel, but not of ClC-3, profoundly reduced the rear than in the lamellipodium (front) of migrating cells migration of monocytes in a Boyden chamber assay, as well [12, 36, 118] or at sites of attachment [97]. The effects of as transendothelial migration of neutrophil granulocytes Ca2+ ions on the cellular migration machinery are manifold, [51, 83]. as outlined above. The requirement of a temporal and 2+ spatial regulation of [Ca ]i during migration demands for a precise tuning of the transport proteins involved in Aquaporins maintaining the intracellular Ca2+ homeostasis. Thus, inhibiting one of the major Ca2+ efflux pathways, the We cited a number of studies that propose (localized) Na+/Ca2+ exchanger, strongly impairs migration [23]. volume changes as the common link between ion channel Similarly, the need for efficient Ca2+ influx pathways is activity and cell migration. However, these studies indisputable. One might therefore argue that any Ca2+ neglected the water movement required for volume changes channel could fulfill this need. However, the task of Ca2+ to occur. This gap was filled recently by demonstrating that channels is much more complex since Ca2+ has to meet water channels have a strong impact on migration, too strict temporal and spatial requirements in migrating cells. [133]. Aquaporins (AQP 1, 4, and 9) are concentrated at the However, the molecular identification of Ca2+ channels leading edge of the lamellipodium of migrating CHO cells, involved in cell migration is still at its beginning. astroglial cells, or neutrophil granulocytes [38, 72, 110]. Mechanosensitive Ca2+ channels mediate Ca2+ influx Thus, they are at the ideal position to participate in the local into migrating keratinocytes or fibroblasts [22, 66, 84]. water and solute uptake at this cell pole since the Na+/H+ When these channels are blocked with Gd3+, migration is Pflugers Arch - Eur J Physiol (2007) 453:421–432 427 impaired. Mechanical tension can be built up in migrating indirect evidence for the involvement of TRPV1 channels cells when the rear part is stuck to the substratum while the in migration of dendritic cells [6]. front is actively protruding. Such a scenario is frequently Members of the TRPM family have also been linked to observed in slowly moving cells in which migration is migration. TRPM2 channels are expressed among others in discontinuous and in which protrusion of the leading edge neutrophil granulocytes. They are activated by ADP-ribose and retraction of the rear part do not occur simultaneously. that is formed intracellularly following stimulation of Traction forces are generated at the front [84], and the neutrophils with chemoattractants such as fMLP or IL- mechanical tension is inversely correlated with the lamelli- 8[40]. The contribution of TRPM2 channels to neutrophil podial extension rate [103]. Activation of mechanosensitive motility is shown by preventing chemotaxis in an IL- Ca2+ influx that is blocked by Gd3+ triggers the retraction 8 gradient when the formation of ADP-ribose is inhibited of the rear part by modulating cytoskeletal contractility, cell [39]. TRPM7 is unique in being both a Ca2+-permeable ion adhesiveness, and cell volume [17, 21, 22, 66]. The channel and a kinase [39, 108]. The function of the kinase molecular identity of mechanosensitive Ca2+ channels in domain has been unknown. However, some recent reports migrating cells is not yet known. However, members of the indicate that it modulates cell adhesion and that it therefore transient receptor potential (TRP) superfamily of ion may also be related to cell migration. The kinase domain channels [88] such as TRPC1 and TRPV4 are attractive belongs to the family of α-kinases and is thus a relative of candidates. TRPC1 channels are activated upon mechanical the Dictyostelium myosin heavy chain kinase [106]. stimulation even when reconstituted in liposomes [76]. Myosin heavy chain kinases play a crucial role in They are also expressed in keratinocytes [13] and have a actomyosin remodeling in Dictyostelium, which is impor- similar conductance as the mechanosensitive channel tant for allowing rapid shape changes during cell migration. shown by Lee et al. [66]. Much of the uncertainty with Ca2+-dependent phosphorylation of myosin heavy chain respect to the molecular identification of mechanosensitive leads to the disassembly of myosin IIA filaments and Ca2+ channels is due to the lack of specific inhibitors. This promotes cell spreading and adhesion [132]. It is now difficulty was circumvented by overexpressing or geneti- shown that these effects can be mimicked by overexpress- cally silencing TRPC1 expression in epithelial cells. ing TRPM7 in neuroblastoma cells. TRPM7 associates with TRPC1 expression correlated with the speed of epithelial the actomyosin cytoskeleton and phosphorylates myosin restitution [102]. TRPC1 channels were also shown to be IIA heavy chain [15]. In another study, different effects of involved in the guidance of nerve growth cones [123, 136]. TRPM7 overexpression were reported. Adhesion and TRPV4 is a mechanosensitive member of the TRPV spreading of HEK293 cells are reduced when TRPM7 is channel family [68]. It is involved in osmoregulation [70]. overexpressed [128]. This is explained by activation of the Recent studies showed that TRPV4 together with TRPV1 Ca2+-dependent phosphatase m-calpain following TRPM7- channels are important Ca2+ influx channels in hepatoblas- mediated Ca2+ influx at peripheral adhesion complexes. toma (HepG2) cells [135]. Ca2+ influx rises when HepG2 Why these two studies, although both pointing to a cells are pretreated with hepatocyte growth factor (HGF). regulatory role of TRPM7 channels for cell adhesion, This effect was most prominent in cells with a migratory yielded opposing results is presently unknown. phenotype. We could now show that activation of TRPV1 Other Ca2+ channels were found to be required for and TRPV4 channels accelerates migration of HepG2 cells migration of neuronal cells that migrate postnatally to their pretreated with HGF. HGF by itself has no effect on final place of work. NMDA receptors and voltage-gated migration [138]. Whereas the stimulatory effect of TRPV4 Ca2+ channels play important, stage-specific roles in channels on migration may be related to their mechano- controlling migration of cortical neurons and granular cells sensitivity, the influence of TRPV1 channels could be due from the developing cerebellum [8, 57, 58] (reviewed in to their interaction with microtubules. TRPV1 channels [56]and[60]). Neurotransmitters like glutamate (and interact with microtubules and thereby stabilize them, GABA) appear to stimulate migration in a paracrine fashion whereas the activation of TRPV1 channels promotes the [74]. N-type Ca2+ channels and NMDA receptors contrib- disassembly of dynamic microtubules [33]. Assuming that ute to the formation of spontaneous intracellular Ca2+ TRPV1 channels, like TRPC1 channels in neutrophils [52], transients whose amplitude and frequency are positively are concentrated at the leading edge of HepG2 cells, correlated with the rate of movement of cerebellar granule activation of TRPV1 would reinforce the asymmetry of cells [59]. Thus, the cessation of these transients leads to microtubules that are more stable at the cell front than at the the termination of migration [62]. Postembryonic neuronal trailing edge [112]. Thus, activating TRPV1 channels could migration that depends on voltage-gated calcium channels modify the microtubule dynamics in a way that allows an was also observed in the nematode Caenorhabditis elegans easier retraction of the rear end and thereby stimulates [130]. Similarly, migration of antennal lobe glial cells and migration. Other studies with TRPV1 −/− mice provided development of mature olfactory glomeruli in Manduca 428 Pflugers Arch - Eur J Physiol (2007) 453:421–432 sexta (tobacco hornworm) require the activity of voltage- channels are also affected by intracellular signaling cascades gated calcium channels [71]. triggered for example by the activation of chemokine receptors. On the other hand, ion channels are essential components of these signaling cascades so that the inhibition Na+ channels of salt and water movement prevents the stimulation with chemokines. Voltage-gated Na+ channels are usually seen in the context One of the challenges for the future is to provide the of excitability of neuronal or muscle cells. However, they proof for the physiological relevance of a given ion channel are also expressed in metastatic tumor cells [29, 31, 32] and for cell migration in an in vivo situation. The availability of in a subpopulation of T lymphocytes [30]. In these cells, a knockout mice and the improvement of intravital micros- correlation between Na+ channel expression and motility/ copy techniques [16] will greatly help achieve this goal. It invasiveness was shown. So far, it is unclear how voltage- would advance the field greatly if for example the gated Na+ channels affect migration. Possible mechanisms contribution of ion channels to localized changes of the include among others the registration of endogenous volume of migrating cells could be demonstrated intravi- electric fields or the effects on the intracellular Na+ tally. So far, localized volume changes had been visualized concentration that alters the extrusion of Ca2+ via the Na+/ only in vitro [114]. The ultimate question will of course be Ca2+ exchanger [85]. whether ion channel blockade can be exploited therapeuti- Acid-sensing ion channels (ASICs) are activated by cally in diseases that are associated with increased extracellular protons and belong to the epithelial Na+ migratory activity such as inflammation and tumor metas- channel/degenerin (ENaC/DEG) superfamily of cation tases or in wound healing. The first promising results have channels. They are expressed primarily in neurons but also been obtained [7, 38, 79, 110, 131, 143]. These studies also in high-grade gliomas [134]. The respective - highlight the need for the development of new and more inhibitable Na+ current is absent in normal astrocytes that specific inhibitors. However, until ion channel blockade can express ASIC1 and ASIC2 in the plasma membrane. be applied therapeutically, much basic work on their function ASIC2 is, however, trapped in the ER/Golgi apparatus of in cell migration has to be done. What is, for example, the high-grade gliomas. This intracellular retention of ASIC2 impact of the local microenvironment within a solid tumor or leads to the activation of an amiloride-inhibitable cation a focus of inflammation on ion channel function and expres- current. Interestingly, the presence of the amiloride-inhib- sion? How are ion channels kept in the plasma membrane itable current correlates with the ability of glioma cells to and targeted to distinct domains in a migrating cell despite migrate. Blocking this current with amiloride also inhibits the fact that the plasma membrane is constantly recycling at a migration [134]. fast rate [2]? Thus, we will experience exciting findings in the years to come that will underscore the importance of ion and water flow for the movement of cells. Conclusion and outlook Acknowledgments The authors greatly acknowledge the contribu- In the past, most of the research on cell migration was tions of former and present members of our laboratory. Work from our focused on topics like cytoskeletal dynamics, cell–matrix laboratory was supported by Deutsche Forschungsgemeinschaft grants SCHW 407/9-1,-2, 407/10-1, RE 1284/2-1, -2, and by the fund interactions, or signaling networks linking extracellular Innovative Medical Research of the University of Münster Medical (chemotactic) stimuli to cytoskeletal dynamics. The contri- School. bution of ion channels to the cellular migration machinery was largely neglected. However, work from the last 10– 15 years provides compelling evidence that ion channels and aquaporins are indispensable for efficient cell migra- References tion. They constitute important signaling molecules in the cell membrane. Some of the channels respond to signals 1. 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