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Kamkin A, Kiseleva I, editors. Mechanosensitivity in Cells and Tissues. Moscow: Academia; 2005.

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Mechanosensitivity in Cells and Tissues.

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Mechanosensitive Cation Channels of Leech Neurons

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Mechanosensitive ion channels are expressed in the central nervous system of the medicinal leech. The plasma membranes of neurons whose primary function is not sensory contain stretch-activated cation channels in relatively high density. Different stimuli, associated with membrane deformation, such as perfusion with hypotonic solutions or pressure pulses applied to the patch pipette, increase the channel's open probability. These cation channels contribute to macroscopic elevations of intracellular free calcium concentration produced by cell mechanical stimulation. Leech cation channels exhibit two activity modes differing both in kinetics and in conductance. In regenerating neurons one of these modes, characterized by long mean channel open time and high mechanosusceptibility, is preferentially expressed. Gentamicin, a voltage-dependent blocker of these channels, affects the neurite growth in culture. A possible role of mechanosensitive cation channels in the processes of neurite growth is discussed.

Introduction

Mechanosensitive ion channels (MS) have been shown to be widely expressed in most cell types [28, 41, 42]. As far as the nervous system is concerned, specific functional roles of MS channels have been demonstrated in hearing, touch, balance, proprioreception and osmoreception [40]. However, it is still unclear why most neurons, which are not involved in sensory mechanoreception, express MS channels. The presence of these channels in nonspecialized neurons can be related to the need for cells to cope with general functions such as volume and electrolyte homeostasis or cell movement regulation. In particular, extending neurites of developing or regenerating neurons migrate through complex environments to reach appropriate target regions and the guidance of nerve fibers to their final destination can be considered as a series of short-range projections under the influence of local cues. Mechanical interaction between cell and substrate is quite important since the nature of the substrate on which growth cones navigate has a powerful effect on the remodelling of the nerve cell processes [17]. Since adhesion and advance of nerve growth cones are associated both with changes in membrane tension [24], and with transient elevations of intracellular calcium ([Ca2+]i) [16], mechanosensitive cation channels may play critical roles in the mechanotransduction response [15, 21]. To better understand the mechanisms by which cells transduce changes in membrane tension into different biochemical responses which regulate growth and differentiation, we should know how environmental factors affect MS channels and how signals generated by MS channels are integrated with other messengers inside the cell. Recent studies have addressed in medicinal leech related issues concerning 1) the ability of MS cation channels to contribute to intracellular calcium homeostasis; 2) the effect of MS blockers on the axonal growth; 3) the functional expression of MS cation channels in quiescent and regenerating neurons; 4) the sensitivity of channel activity to intracellular signals; 5) the modulation of channel activity by phosphorrylation/dephosphorylation processes.

In this review the findings in the physiology of leech cation channels will be summarized and the possible role of these channels in quiescent and regenerating neurons will be discussed.

Large conductance stretch-activated channels of leech neurons

Ion selectivity and single channel conductance

Leech stretch-activated channels (SACs) were identified with the use of patch-clamp recording techniques in symmetrical high K+ (120 mM) solutions [36]. Both on-cell and excised patches displayed large amplitude unitary currents, sensitive to negative pressure applied to the pipette. The current-voltage relation was not linear, but exhibited an outward rectification. The single channel slope conductance, computed at depolarizing membrane potentials, was about 200 pS.

Ion selectivity was assessed by experiments of ion substitution. The current-voltage plot did not change after substitution of 50% of chloride ions with acetate ions in the bath, whereas the reversal potential of the single channel current shifted towards the theoretical potassium equilibrium potential when K+ concentration in the bath was changed. The cation selectivity was confirmed by experiments of partial or total substitution of K+ with Na+. Sodium ions can pass through the SACs; however the single-channel currents in symmetrical Na+ are about 70% of those measured in symmetrical K+ solutions.

The ability of Ca2+ to carry current through leech SACs was demonstrated in different ways. Single channel activity was recorded from inside-out patches by filling the pipette tip with 120 mM KCl and backfilling with 110 mM CaCl2, according to the technique described by Auerbach [1]. This approach allowed us to unambiguously identify the SAC unitary currents carried by K+ at the beginning of the recording and to follow their changes in amplitude while Ca2+ was diffusing to the electrode tip. In the chosen experimental conditions, the time taken by Ca2+ to nearly completely substitute K+ was about 20 minutes and the current amplitude decreased to reach a steady state value corresponding to about 65 pS. Similar values of conductance were measured when SACs were studied in symmetrical high calcium (110 mM) solutions [5].

Recently, the contribution of SACs to the intracellular calcium homeostasis was measured by fura-2 imaging. Cell bodies of identified leech neurons were mechanically isolated, plated onto concanavalin A and maintained in culture for a few days. After loading fura-2 AM into cells, ratiometric measurements of intracellular free calcium in single cell bodies were carried out, while transiently perfusing with hypotonic solution, at a physiological external calcium concentration (1.8 mM). Macroscopic reversible [Ca2+]i elevations were associated with hypotonic cell swelling (Fig. 1).

Figure 1. Fura-2 measurements of [Ca2+]i (red trace) in a leech AP neuron at rest, as it swells in hypotonic solution and as it reshrinks.

Figure 1

Fura-2 measurements of [Ca2+]i (red trace) in a leech AP neuron at rest, as it swells in hypotonic solution and as it reshrinks. Normalized values of membrane area are plotted (blue trace) Representative ratio images (340/380 nm) at the times marked a, (more...)

Two components contribute to this response. One is independent of external calcium and is presumably due to the ion release from internal stores, the other component is sensitive to Gd+3 and to gentamicin.

A partial contribution of SACs to the calcium signal can be revealed by inducing cell swelling in the presence of a SAC blocker (either Gd+3 or gentamicin) and then withdrawing it. Fig. 2 illustrates how removal of the blocker induces an increase of calcium signal. Changing the order of presentation of the stimuli, first hypotonic solution without channel blocker and then addition of the blocker, induced a reduction of the calcium signal [unpublished results].

Figure 2. Measurements of [Ca2+]i (red trace) and membrane area (blue trace) at rest, during hypotonic swelling in the presence of 10 μM Gd+3 (+bl) and after removal of the blocker (-bl).

Figure 2

Measurements of [Ca2+]i (red trace) and membrane area (blue trace) at rest, during hypotonic swelling in the presence of 10 μM Gd+3 (+bl) and after removal of the blocker (-bl). Representative ratio images at the times marked a, b and c are displayed. (more...)

Sensitivity to blockers

The lack of suitable compounds which selectively block MS channels prompted us to use a panel of blockers to identify MS channels. Leech SACs are sensitive to the extracellular application of the three main non specific blockers of mechanogated cation channels [18]. Gd+3 induces a fast flickery voltage-independent block of the single channel activity. Amiloride causes a partial and voltage-dependent block. Gentamicin does not significantly affect the single channel current at positive membrane potentials, whereas at negative potentials it completely blocks the pore [5]. This aminoglycoside, unlike Gd+3, is unable to block voltage-dependent Ca2+ channels of leech neurons at a dose of 200 μM, which completely blocks SACs. Gentamicin has been routinely used as an antibiotic in leech cell culture media. Consequently, it has received the most attention in designing experiments to provide evidence for implicating SACs in cellular growth processes.

Activation mechanisms

Application of slow negative pressure to the patch pipette performs a rough stimulation of leech SACs [36]. To overcome the limitation due to the slow rate of change of applied pressure, SAC activity was recently analysed by applying to the patch pipette fast pressure changes, characterized by a 20% to 80% step response time less than 1 ms. The experimental device, illustrated in Fig. 3 A and B, was built according to Hurwitz and Segal [20]. Fast channel activation (latency of channel turn-on 8 ms) and deactivation were observed (Fig. 3C), in keeping with the mean criterion suggested for a direct mechanism of mechanosensitivity [18].

Figure 3. Image (A) and schematic diagram (B) of the pressure step system.

Figure 3

Image (A) and schematic diagram (B) of the pressure step system. EV1 and EV2: solenoid valves; T: piezoresistive pressure transducer; H: pipette holder; D: tap; M: microchamber. A SAC response (upper trace) to a pulse of –50 mmHg (lower trace), (more...)

Leech SACs can be activated in cell-attached configuration, which is supposed to maintain the main relationships between the channel molecule and both cytosol and cytoskeleton. Slow hypotonic perfusion of the whole preparation induces cell swelling and parallel channel activation [36]. Fura-2 imaging showed that hypotonic cell swelling associated with about 10% increase of cell surface was followed by reversible elevations of intracellular calcium concentration and only part of this response was blocked by Gd3+ or gentamicin (unpublished results).

In a series of papers, it has been shown that membrane depolarization slowly activates mechanogated channels in Xenopus oocytes, through membrane movements that resulted in tension changes [10]. Intriguing aspects of this type of activation are its dependence on the hard glass used for pipette fabrication and its absence in outside-out configuration. Although the features of voltage activation of mechanosensitive channels in Xenopus oocytes previously reported [9, 10, 43] were not entirely confirmed [49], this phenomenon was observed in SACs of leech; membrane patches excised from AP neurons and held at a positive membrane potential displayed only occasional brief openings. In the absence of applied pressure a progressive slow activation developed with time and alternate application of hyperpolarizing and depolarizing steps of tens of seconds induced channel deactivation and activation, respectively, with extremely slow kinetics [29]. Results obtained in leech differ in two relevant aspects from those in Xenopus because the phenomenon is observed in both inside-out and outside-out configurations as well as with pipettes fabricated with soft glass. However some findings seem to confirm that membrane potential affects SACs through changes of membrane tension. On one hand, modifying the basal membrane tension affected the concurrent voltage-induced channel activation; the application of negative pressure to the pipette, during voltage-induced activation, enhanced, whereas positive pressure reduced, channel activity. On the other hand, the delay of voltage-induced activation was widely variable from patch to patch but inversely related to the mean level of channel activity. This result was expected, assuming that basal channel activity is a measure of the resting tension in the membrane patch. These findings are consistent with the conclusion that the observed voltage dependence of SACs is associated with voltage-induced changes in membrane tension. In leech the effect of membrane depolarization was found equivalent to application of negative pressure to the pipette (convex curving) [29]. This is in agreement with the observations in Xenopus oocytes reported by Silberberg and Magleby [43].

A direct quantitative evaluation of voltage-induced membrane movements was made by Zhang et al. [51], who studied the converse flexoelectric effect [30, 37] by combining atomic force microscopy with whole-cell patch clamp. Depolarization was found to move up the cantilever and the peak displacement was found to be linear with voltage. These results put on firm experimental ground the thermodynamical prediction of this phenomenon.

SACs in regenerating neurons

The basic strategies and molecular mechanisms by which neuronal projections are established are strongly conserved. Accordingly, the large growth cones of cultured neurons of some invertebrates have been extensively exploited to understand the intrinsic mechanisms of growth [12, 14, 17]. The leech nervous system has proved to be suitable for studies of nerve regeneration because characterized neurons can restore their synaptic connections accurately after injury of their axons [31]. The outgrowth of neurites by single leech neurons in culture is markedly affected by the substrate [17]. The axon growth is slow and delayed on the poly(L)-lysine, fast and extensive on the plant lectin concanavalin A, straight and with few branches on extracellular matrix (ECM) extracts. Also the growth cone morphology reflects its interactions with substrate. Growth cones are large and complex or small and club-shaped when leech neurons are plated on concanavalin A or ECM extracts, respectively. The growth cones of leech neurons which are not mechanosensory, such as AP cells, contain SACs in high density [5]. Although often the open probability of SACs from growth cones is high, even in the absence of applied pressure to the patch electrode, they are sensitive to stretch both in cell-attached and in inside-out configuration.

The effects of blocking SACs on neurite outgrowth were studied by time-lapse video recording on cultured AP neurons. The blocker selected for this study was gentamicin. Phase contrast images were collected at 5-min intervals, between 10 and 40 hours after plating of isolated cells onto concanavalin A, with or without gentamicin in the culture medium. Each image of neurite arborization was processed to produce binary and single pixel replicas, used to compute some morphometric indicators, such as total length and density of arborization as well as mean caliber and mean length of branches. Gentamicin was found to induce a transient increase in the number of segments of the same mean length, between 10 and 20 hours after plating [5].

Activity modes

One of the main arguments against the role of SACs as mechanotransducers in non-specialized cells is the low mechanosusceptibility found in some preparations [46]. Although SACs exhibit intrinsic sensitivity to mechanical stimuli, as shown by their prompt activation in membrane blebs lacking a cortical cytoskeleton [48], extrinsic factors such as membrane infolding [49, 50] and submembrane shock absorber cytoskeletal structures can reduce the fraction of mechanical energy forcing the channels [23]. The attractive hypothesis that SACs might exhibit different mechanosusceptibility as a consequence of facing different microenvironmental conditions has been put forward [40, 46]. From this standpoint, the functional properties of the same mechanosensitive cation channels of leech AP neurons were comparatively studied in cell bodies of freshly desheathed ganglia and in cell bodies and growth cones of their counterparts in culture. The rationale of this study was to detect possible differences of SACs expressed by quiescent and growing neurons. The results revealed clear-cut differences in the activity pattern of SACs from quiescent and regenerating neurons. Two distinctive activity modes named spike-like (SL) and multiconductance (MC) could be characterized [35]. Single SAC activity in inside-out patches excised from freshly desheated cell bodies typically consisted of bursts of activity with transition frequencies of up to tens of Hz and dwell times so brief as to be attenuated by the filter cutoff. The all points histogram showed a single open level corresponding to 115 pS in symmetrical Na+ solutions. The mean channel open time never exceeded 10 ms and the distributions of the open times could be fitted by the sum of two exponentials, with time constants shorter than 2 and 10 ms (Fig. 4 upper panels). Conversely, in the same experimental conditions, current traces from inside-out patches isolated from neurons growing in culture displayed a typical pattern consisting of long openings occurring at frequencies lower than 5 Hz. Open current fluctuated between a number of distinct levels. The all points histogram presented well defined peaks, indicating the presence of a main level corresponding to 115 pS, conductance sublevels corresponding to about 40, and 80 pS, along with a superlevel of 150 pS. The mean channel open time exceeded 200 ms and the exponentials fitting the distribution of open dwell times had time constants of about 15 and 150 ms (Fig. 4 lower panels). A further outcome was that the percentage of time spent at the 80 pS subconductance level was significantly higher in patches isolated from growth cones than in those from cell bodies of cultured neurons.

Figure 4. Activity modes of single SACs in inside-out patches excised from a freshly desheated cell body (upper panels) and from a neuron growing in culture (lower panels).

Figure 4

Activity modes of single SACs in inside-out patches excised from a freshly desheated cell body (upper panels) and from a neuron growing in culture (lower panels). Current records (A), all points histograms (B) and open dwell time distributions (C) are (more...)

It is likely that these modes belong to the same ion channel because they share main conductance, outward rectification, activation by stretch and slow activation by voltage-induced changes in membrane tension. Furthermore, they share the sensitivity to the blocking agents Gd+3 and gentamicin, together with the specific type of blocking.

The occurrence of two activity modes might have a physiological meaning or simply reveal a different response to excision. They do not seem to be associated with soluble modulating factors because of the absence of rundown phenomena. One of the simplest hypotheses to account for the two modes is that the assembly of membrane-cytoskeleton complex of cultured neurons, affected by cell adhesion and growth, might be favourable to MC mode and to 80 pS conductance level. However a different composition in subunits cannot be ruled out.

Features of SACs in membrane patches from cultured leech neurons reveal distinctive susceptibility to membrane potential, with short delays to voltage-induced channel activation and high sensitivity to small changes of holding membrane potential (even 20 mV). This enhanced voltage sensitivity may be indicative of a high meccanosusceptibility of SACs in culture and in particular from growth cones. A similar result has been reported for SA K+ channels of molluscan neurons [44].

Modulation

The ubiquitous distribution of SACs in leech neurons makes cell membranes able to transform mechanical stimuli into cation fluxes. In particular, these channels can positively contribute to internal calcium homeostasis, either directly, because of their high calcium conductance, and indirectly, by increasing the open probability of voltage-dependent calcium channels, due to membrane depolarization. As expected, SAC activity must be strongly controlled to avoid cell calcium loading. Various mechanoprotective mechanisms can be activated by different cell types to limit the deformability of the membrane and to desensitize SACs. Recruitment of subcortical actin to sites of membrane deformation [19] as well as exocytic fusion of vescicles provide feedback responses to mechanical stimuli [11, 23].

Cytochalasin D treatment of inside-out patches from leech neurons was found to be effective in activating SACs [35]. This effect takes minutes to develop and might be conceived as due to a redistribution of tensile forces between membrane and cytoskeleton. Interestingly, intracellular calcium concentration strongly modulates SAC activity. In the absence of applied pressure, the perfusion of the inner face of inside-out patches with millimolar calcium consistently activated SACs within minutes, but channel activity was reversibly depressed by lowering calcium concentration at 0.1 μM [35]. The long delays to activation make direct effects of calcium unlikely, suggesting alternative mechanisms. Calcium influx disrupts microfilaments in growth cones of leech AP neurons [32] and the effect was probably due to the calcium sensitivity of enzymes promoting actin depolymerization [38]. Both cytochalasin D and high calcium effects might be due to changes in membrane tension, secondary to cytoskeletal rearrangements. The notion that the function of ion channels is affected by actin is well established. Actin depolymerization underlies NMDA rundown in hippocampal neurons [39] and actin filament organization modulates epithelial Na+ channels [7] as well as Cl- channels involved in morphological changes of astrocytes [25]. As far as mechanosensitive channels are concerned, mechanotransduction in human fibroblasts has been found to be regulated by actin network [47] and mechanosensitivity of SA K+ channels of Lymnaea neurons is enhanced by actin depolymerization [46].

On a faster time scale, ongoing modulation of leech SACs by environmental factors was recently found. In the absence of applied pressure, channel activity was affected by pH. Fig. 5 illustrates the effect of six changes of pH on the mean current and opening frequency that were reversibly increased by internal acidification. A robust upmodulation of channel activity was observed by perfusion of the inner face of inside-out membrane patches with MgATP. Fig. 6 illustrates this modulation. Channel activity which was extremely low before ATP addition, appeared upon ATP perfusion to run down after wash-out and to reappear after ATP readmission. Consistently, the time lag between the addition of the stimulating solution and the initial change of channel activity was very short whereas the deactivation was slow, even at doses as low as 50 μM. Control experiments demonstrated that ATP did not act directly as a ligand but this modulation required ATP hydrolysis. Experiments are in progress to determine the nature of the enzymes involved in this control as well as the target molecule.

Figure 5. Modulation of SACs by pHi.

Figure 5

Modulation of SACs by pHi. Samples of activity recorded from an inside-out patch membrane during perfusion with solutions at different pH and plot of mean current calculated from consecutive 1-s-long data segments. Outward currents are displayed as upward transitions. (more...)

Figure 6. Reversible up-modulation of SAC activity by MgATP.

Figure 6

Reversible up-modulation of SAC activity by MgATP. Outward currents are displayed as upward transitions. The tick on the right of records indicates the closed channel level. Vm = +80 mV. Filtering 1 KHz.

Possible physiological roles of SACs in leech neurons

SACs were found in non sensory neurons such as AP and Retzius cells as well as in primary mechanosensory neurons T, P and N. Although no direct evidence is available to assign to SAC the physiological role of mechanotransducer channel, some functional properties of these channels make them good candidates. On one hand, fast activation and deactivation would allow cell membranes to transduce mechanical deformations on a quick time scale. On the other hand, cationic selectivity and pharmacological features are similar to those of TRP-like channels of vertebrates' hair cells [22]. T, P and N are first-order sensory cells, responding selectively to touch, pressure or noxious mechanical stimulation of the skin surface. Nicholls and Baylor [33] demonstrated that low-threshold T cells respond with transient bursts of impulses to light touch, whereas activation of P and N cells, consisting in prolonged discharges, occurs when stronger maintained pressure or pinching is applied to the skin. The lack of intrinsic rapid adaptation makes SACs suitable to transduce both touch and pressure sensory modalities.

The morphology and the distribution of touch and nociceptive terminals have been studied in details in leech and landmarks of cutaneous receptive fields have been described [2, 3]. More recently, stretch sensory neurons innervating the body wall muscle have been identified in leech. However, unlike cutaneous mechanosensory neurons, muscle stretch receptors respond with hyperpolarizing membrane potentials to muscle stretch and do not display adaptation [4]. SAC properties do not appear to fit the requirements for the source of this generator current. The response of muscle receptors to stretch needs the action of stretch-activated K+ channels or of stretch-inactivated cation channels (SICs). Unfortunately, at present no evidence of SICs in leech is available.

As already pointed out [41], the physiological function of mechano-sensitive channels in cells whose function is not sensory remains to be determined. The same question arises for AP or Retzius cell in leech. The ubiquitous distribution of SACs in leech neurons suggests that these channels participate in some general functions, such as volume and osmotic homeostasis in quiescent neurons. Since it is supposed that cell swelling activate most SACs in a cell, the physiological response can be performed by channels with low mechanosusceptibility, probably due to links between membrane and subcortical actin framework [23]. This mechanoprotection prevents cell calcium loading by mechanical stress in a highly extensible body, lacking a rigid skeleton. In keeping with this view, in the cell bodies of quiescent neurons SACs mainly display the SL mode, characterized by low mean channel open time. Cultured neurons regenerate their processes and hence increase two additional cell activities: adhesion and movement, both associated with remarkable cytoskeletal reorganization. Most SACs in regenerating neurons exhibit the MC mode, with long mean channel open time and spontaneous activity.

Voltage-sensitivity of leech SACs was likely due to changes in membrane tension [10, 51], but, unlike pressure stimuli, electrical stimuli did not alter channel response with time. Thus, depolarization was used as a tool to test the channel mechanosusceptibility. An exciting outcome was a clear-cut difference in latency to voltage activation between patches from quiescent cells and those from regenerating neurons [29]. The enhanced voltage sensitivity may reveal a high mechanosusceptibility of leech SA cation channels in regenerating neurons and in growth cones, similar to that reported for SA K+ channels in molluscan neurons [44]. An attracting working hypothesis grounded on the ubiquitous distribution of SACs in leech neurons as well as on the functional differences observed in quiescent and regenerating neurons, is to conceive that SACs have functional roles which depend on the specific environment in which they operate. In particular, the high spontaneous activity of SACs excised from growth cones and their high susceptibility to voltage-induced membrane tension, suggest that in this cellular region the efficacy of mechanoprotection by the cytoskeletal network is probably reduced and/or local factors enhance channel activity.

The dependence of SAC activity on intracellular Ca2+ and actin enables these channels to distinguish between passive mechanical stress and active movements associated with rearrangements of cortical cytoskeleton. During active movements, the channel mechanosusceptibility might be greatly enhanced by increased concentration of intracellular Ca2+, by actin depolymerization, as well as by signal transduction pathways stimulated by dynamic growth cone-substrate interaction.

Different classes of growth cones share the property of generating periodic elevations of [Ca2+]i as they migrate in vivo. Naturally occurring Ca2+ transients were shown to regulate growth cone extension [13, 16] and the rate of axon outgrowth is inversely proportional to the frequency of calcium transients, which appear as a natural signalling mechanism that regulates the axon extension [16]. Ca2+-induced Ca2+ release is mainly responsible for the calcium wave amplitude; however, influx through plasma membrane channels is a significant contributor to the transients. On the other hand, there is a wide consensus that neurite and growth cones membranes experience large changes in tension during their extension and retraction [24]. Ca2+ can carry current through leech SACs and a contribution of these channels to intracellular calcium homeostasis has been found. Thus, stretch-sensitive cation channels of neuronal growth cones, unevenly activated, could provide a local and contingent link between membrane tension and transmembrane Ca2+ fluxes, as found in other cells [26].

The sensitivity to gentamicin of the axon outgrowth in AP cells is consistent with this hypothesis, although the transient nature of this sensitivity cannot be easily explained. However, the function of SACs does not seem critical for cell growth, as indicated by the occurrence of growth in the presence of gentamicin. Other channels, such as voltage-gated calcium channels [6, 27], can be involved in cell mechanotransduction.

Although preliminary, results of channel modulation indicate that leech SACs share some properties of 2P/4TMS TREK/TRAAK channels of mammalian neurons. Both types are mechanosensitive, sensitive to voltage, to intracellular pH and to intracellular ATP. Their activity seems to result from an integration of different environmental signals, stretch included.

Conclusions and perspectives

A major advantage in studying MS channels in different cells is that a screening for common features can be made. Neuronal SACs of medicinal leech share the ion selectivity and pharmacological properties of typical MS cation channels such as those of Xenopus oocytes and vertebrate hair cells. Moreover, they also exhibit a voltage-sensitivity similar to that first described for Xenopus cation channels, but without the technical constraints found in oocytes. The physiological relevance of voltage-induced membrane movements in special regions, such as growth cones, remains to be determined, as recently suggested [51].

Our knowledge regarding the activation of leech SACs is still emerging and needs to be improved. However, the available data concerning their polymodal activation by physical and chemical stimuli make these channels very similar to the mammalian K+ channels, structurally characterized by the presence of a tandem of P domains. Recent studies with recombinant channels revealed that the activity of mechanosensitive TREK and TRAAK channels, expressed in neurons, is modulated by an unusual variety of environmental stimuli [34]. The general idea that MS channel sensitivity is under local regulation has been put forward by various authors [8, 40, 41, 45]. In particular this control is probably complex at the tips of extending neurites, where the environment is likely to confront the growth cone with a variety of simultaneous influences and the growth cone must therefore integrate inputs and choose an appropriate final response.

On the other hand, each preparation allows one to focus on peculiar aspects of the channel expression in a specific cell type. One of the most interesting outcomes of the research on leech SACs is the alternative expression of two clear-cut activity modes. Further studies will concentrate on defining whether experimental manipulations of cytoskeleton can induce transitions between the two modes or can change the times spent at different conductance levels.

It will be also important to carry out experiments addressing the modulation of SACs by phosphorylation/dephosphorylation processes, with special attention to protein kinases which have been found to affect cytoprotective responses to stretching [11].

Acknowledgement

The authors gratefully acknowledge Dr. Rozenne Guegan and Dr. Debora Ricci for their participation to preliminary experiments of calcium imaging and P. Orsini, F. Montanari and E. Cardaci for their expert technical assistance. The work in the authors' laboratories has been financially supported by grants from MURST and CNR.

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