Role of the actin cytoskeleton on epithelial Na

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1 Kidney International, Vol. 48 (1995), pp Role of the atin ytoskeleton on epithelial Na hannel regulation Hoiio F. ANTIELLO Renal Unit, Massahusetts General Hospital and Department of Mediine, Harvard Medial Shoo4 Gharlestown, Massahusetts, USA Role of the atin ytoskeleton on epithelial Na hannel regulation. The regulatory role of atin filament organization on epithelial Na hannel ativity is reviewed in this report. The atin ytoskeleton, onsisting of atin filaments and assoiated atin-binding proteins, is essential to various ellular events inluding the maintenane of ell shape, the onset of ell motility, and the distribution and stability of integral membrane proteins. Funtional interations between the atin ytoskelton and speifi membrane transport proteins are, howver, not as well understood. Reent studies from our laboratory have determined that dynami hanges in the atin ytoskeletal organization may represent a novel signaling mehanism in the regulation of ion transport in epithelia. This report summarizes work onduted in our laboratory leading to an understanding of the moleular steps assoiated with the regulatory role of the atin-based ytoskeleton on epithelial Na hannel funtion. The basis of this interation lies on the regulation by atin-binding proteins and adjaent strutures, of atin filament organization whih in turn, modulates ion hannel ativity. The sope of this interation may extend to suh relevant ellular events as the vasopressin response in the kidney. The atin-based ytoskeleton, onsisting of atin filaments and assoiated proteins, is a dynami struture whih plays an essential role in the regulation of ellular events inluding the stability of ell shape and the onset of ell motility [1], the distribution of integral membrane proteins [2, 3] and the ontrol of hormone ation [41. The role of atin interations with other strutures inluding ion hannels, however, is largely unknown. Atin and the various atin-binding proteins ouple to a variety of transmembrane proteins inluding most ion transport moleules [2, 5]. Ankyrin and spetrin, for example, o-loalize with the band 3 anion exhanger [6], the a subunit of the Na F,K±ATPase in epithelial ells [7], and the rat brain, voltage-sensitive Na hannels [8]. Other ion transport proteins inluding the Na,K,I -otranporter, and the Na IH exhanger are also linked to ytoskeletal omponents [9, 10]. More reent findings also indiate that the Na hannel omplex of epithelial A6 ells o-purifies with ankyrin, fodrin, and atin itself [111. Funtional interations between the atin ytoskeleton and ion hannels have been also reently reported for a2 hannels from neurons [12, 13]. These data are onsistent with the reent observation that atin filaments o-loalize and are apable of modulating Na hannel ativity in epithelial ells [14]. Under physiologial onditions inluding high ioni strength and suh substrates as ATP, for example, atin readily polymer by the International Soiety of Nephrology izes into long helial (rigid) filaments of several rm in length [15]. One polymerized, filamentous (F) atin remains as an a helix in dynami equilibrium with monomeri (G) atin (Fig. 1). Only polymerized atin is known to have a biologial role. ontroversy still surrounds the average length of physiologially relevant atin filaments [16]. Atin is also known to bind and slowly hydrolyze ATP, whereby a phosphate and ADP are non-ovalently attahed to the protein in a proess that may entail hanges in the mehanial stability of the polymer [17]. The Mg2-ATP omplex binds to, and hanges the onformation of G-atin, thus inreasing the affinity of atin monomers to nuleate and form oligomers. Interestingly, this phenomenon an be mimiked by the fungal toxins, ytohalasins, whih have a nuleating effet upon binding to the atin monomer [18, 19]. Mg2-ATP-bound G-atin is omplexed into F-atin in a multistep proess whih inludes short polymeri forms (nuleated atin), and longer forms generated by annealing to finally beome long filaments [20, 21]. A large number of proteins that modulate atin filament organization has been desribed [22]. These proteins, generally known as atin-binding proteins, will bind to and interat with the various forms of atin in a highly speifi manner. Thus, atinbinding proteins an bind to either G- or F-atin, therefore regulating atin polymerization, or may help link atin filaments to other intraellular strutures inluding mirotubules [23], and the plasma membrane [24, 25]. Atin-binding proteins are lassified by their in vitro funtion. Among the G-atin-binding proteins is the group known as profihins, low moleular weight (13 to 15 kd) ytoplasmi proteins, whih bind and stabilize the atin monomer, therefore preventing atin polymerization [26, 27]. The panreati enzyme deoxyribonulease I (DNAse I), a 33 kd protein, also binds G-atin into a omplex with a stoihiometry of 1:1 whih is unable to polymerize [28]. The F-atin-hinding proteins are known to have at least two atin-binding domains and thus indue either the bundling or the ross linking of atin filaments into tridimensional atin filament networks with gel behavior [29]. Depending on the distane between the atinbinding domains, these proteins may enable either the bundling of atin (tightly paked arrays of filaments), as is observed in the omplexes of F-atin found in mirovilli, or otherwise the ross linking of atin [301. Two of the most important proteins in this group are filamin and its homologue atin-binding protein (ABP), whih was first isolated from rabbit marophages [31, 321. Reent studies have determined that filamin also helps atin filaments interat with membrane glyoproteins, a funtion whih is distint from its atin filament ross linking apability [33]. 970

2 antiello: Atin regulates epithelial Na hannels 971 Mg2/ATP atin monomers 0 (G-atin) 0000 DNAse a di o0_ fragmentedd i3elsolin gels (F-atin) Fig. 1. Dynamis of interations among the various onformations of atin. In the presene of MgATP, monomeri (G) atin (Left) readily nuleates into short atin filaments (tn-, tetramers, enter) whih then anneal to produe long atin filaments (Right). The protein DNAse I interats with G-atin thereby inhibiting its polymerization. Intraellular proteins suh as gelsolin ap and/or sever atin filaments, therefore dereasing their visosity. ross linking proteins suh as filamin indue the gelation of the atin filaments. Atinbundling proteins, inluding a-atinin, enable a tightly paked F-atin network or bundle. Two general lasses of proteins an be funtionally distinguished, those that ap and sever atin filaments and proteins that only indue the apping of the barbed end of atin [34]. The apping or severing proteins help fragment the already polymerized atin filaments. The severing effet on the atin filaments is produed when the apping protein seletively binds to one of the ends of the polymer inhibiting the rate of polymerization at that end. Therefore, atin-apping proteins have a similar effet to that of ytohalasins on atin filament organization [35, 36]. One important member of this family is gelsolin, a 90 kd protein originally purified from marophages [37, 38], whose effet on atin filament networks is a2-dependent. Experimental evidene for atin regulation of epithelial Na hannels Role of atin on Na + hannel regulation As indiated above, atin and its assoiated proteins are struturally linked to ion transport proteins. It is thus possible that atin filament organization maybe also a regulatory omponent of membrane transport inluding ion hannel ativity. In this regard, a funtional relationship between atin filament organization and streth-ativated hannels has been long reognized [39 41]. It was originally onluded from experiments entailing the prolonged use of ytohalasins [39] that streth-ativation of ion hannels was mediated by hanges in the ytoplasmi surfae tension maintained through the elasti ativity of the ortial ytoskeleton. We have, on the other hand, proposed a different hypothesis whih is based upon the assumption that hanges in atin filament organization may lend themselves responsible for ion hannel regulation. This interation may be either related to a diret oupling of ytoskeletal omponents to transport proteins and/or the sequential interation of atin filaments with apially loated regulatory pathways. The studies summarized in this report indiate that both possibilities are feasible. The hypothesis that the atin ytoskeleton was involved in the regulation of epithelial Na hannels was first supported by immuno-oloalization studies showing that Na hannels appearing on the ell surfae are always in lose proximity to atin filaments [11, 14]. The studies desribed herein were onduted in the polarized epithelial ell line A6, originated from the distal amphibian nephron, and whih has been extensively studied by us [14, 42 44] and others [45 47], thus providing a useful model for the haraterization of ion transport phenomena in hormonallytargeted renal epithelia. A6 ells at all stages of growth inluding dividing or newlyplated ells showed Na hannels first appearing on mirovilli and apial surfae ridges in lose proximity to atiri filaments whih penetrate to the same points on the ell surfae. This finding raised the possibility that a potential Na hannel-atin filament interation might be an early feature of epithelial ell development. Albeit this interation may serve as a means to ontrol the spatial distribution of Na hannels in the apial membrane of epithelial ells, whih is a requirement of polarized epithelia, the possibility also existed that the o-loalization of atin filaments with the apial Na hannels would also be of a funtional nature. This indeed proved to be the ase. The studies foused on the amilonide-sensitive Na hannels expressed by subonfluent A6 epithelial ells. Under ulture and growth onditions as previously reported [42], this pool of apial Na hannels represents the major pathway for Na reabsorption by this epithelium [48]. Briefly, this amiloride-sensitive Na4 hannel (K1 i0 M) has a Na:K permseletivity of 5 to 1 and a single, non-retifying single hannel ondutane of 8 to 10 ps [14, 42]. The details of the methodology have been previously desribed in the respetive studies. Effet of ytohalasin D treatment on Na hannel ativity. To assess the funtional role of endogenous ortial atin networks on apial Na hannel ativity in A6 ells, atin filament organization was first modified with ytohalasin D (D), a fungal toxin known to depolymerize atin filaments [18, 49]. Ion hannel ativity was followed by the path-lamp tehnique under ellattahed onditions. As expeted from its depolymerizing effet on F-atin, D indued Na hannel ativity within five minutes of addition (Fig. 2). The effet was speifi for D sine other ytohalasins inluding B, A, and E did not eliit ion hannel ativation in either ell-attahed or exised pathes [14]. Beause DNase I also inhibited the D-indued Na hannel ativity, the

3 972 antiello: Atin regulates epithelial Na + hannels A +D ontrol -- Vh=30 mv 18 mm after 5 jig/mi D addition Daddon 'rr'tr F'nnh11'.'...' 1 pa B se 2.0 0z Time, minutes Fig. 2. Effet of ytohalasin D on Na hannel ativation in A6 ells. ells were exposed for various times to ytohalasin D (D, 5 jig/mi) and pathed under ell-attahed onditions. A. Representative traings of ell-attahed pathes under ontrol onditions with no hannel ativity (top traing), and after 18 minutes (middle traing), and 60 minutes (bottom traing) of D addition. NaI (115 mm) was in the pipette and bathing solutions. B. The produt of the mean open probability and the apparent number of hannels is indiated on the ordinate for various times after ytohalasin D addition. Ativation of Na hannels was observed by two mm after addition of ytohalasin D and was ompletely absent after 40 minutes. The data are the mean SM, N 10. Reprodued from [44] with permission. data allowed speulation that short atin filaments might be involved in effeting ion hannel ativation. The possibility that Na hannel ativation triggered by D was indeed mediated by hanges in atin filament organization was further supported by the fat that the D effet was transient and was only observed in ells treated with the drug for periods of time shorter than 40 minutes. However, longer exposures to D resulted in dramati hanges in ell shape and loss of plasma membrane interations

4 antiello: Atin regulates epithelial Na hannels 973 Vh =60 mv ontrol E2 ::LLJti: 21 ii 2 mm after ATP addition [LPA 0.5 se! Fig. 3. Effet of atin and ATP on Na hannel ativity. Addition of atin (5 jsm) to the ytoplasmi side of exised pathes with no spontaneous Na hannels (top traing) indued Na hannel ativity within five minutes (middle traing, representative of 11 experiments). Further addition of ATP (1 mm) signifiantly enhaned the effet of atin alone (bottom traing, representative of 11 experiments). Holding potential was 60 mv in symmetri Na solutions (NaI, extraellular surfae; Na isethionate, ytoplasmi surfae). Reprodued with permission from [14]. with the ytoskeleton. Under these onditions Na hannel ativation was no longer observed [44]. Effet of atin on Na hannel ativity. To further demonstrate that the effet of D on Na hannel ativity was assoiated with hanges in atin filament organization and not in membrane struture as has been previously suggested [39, 50], the effet of exogenous atin was diretly assessed. Addition of purified atin at the ritial onentration for polymerization (>5.0 M) to the ytoplasmi side of exised inside-out pathes, indued and/or inreased Na hannel ativity (Fig. 3) [14]. This would therefore be onsistent with the above results in whih endogenous filament disruption would enable more free soluble atin. The apparent hannel number doubled under these onditions, but no hange was observed in the mean open time of ative Na hannels. This partiular finding indiates that the otherwise quiesent (prior to addition of atin) Na hannels were already present in the path. In ontrast, whenever atin was added after being polymerized to ahieve predominantly long filaments, no Na hannel ativation was observed. Sine atin rapidly polymerizes into short filaments at the onentration and high ioni strength onditions of the bathing solution, it was speulated that short polymers instead of monomeri atin indued hannel ativity. Thus, the nature of atin filament length would be an important parameter on its effet(s) on Na hannel ativation. This hypothesis was further explored by assessing the effet of ATP and atin-binding proteins on the atin-mediated regulation of epithelial Na hannels. ATP helps the nuleation and polymerization of atin by stabilizing its polymeri onformation [17, 20]. Thus the effet of ATP on the atin-indued Na hannel ativity would help evaluate the role of atin filaments on the ativation proess. Addition of ATP (1 mm) to the path-bathing solution ontaining exogenous atin further inreased the average open hannel number (Fig. 3). Interestingly, the addition of ATP also inreased the mean open time of the atin-indued hannels, an indiation that the atin onformation may be important in the onformational hanges that trigger Na hannel ativation. This was further supported by the finding that ADP was without effet on the atin ativation of Na hannels. Addition of ATP to quiesent pathes in the absene of atin was also without effet. Thus, the effet of the nuleotide is onsistent with onformational hanges enabled by the MgATP omplex onto the atin monomer and not prepoly merized (endogenous) atin. The effet of D on the exogenous, atin-mediated Na hannel ativation was also in agreement with this hypothesis, sine addition of the drug after hannel ativation with atin also further inreased the mean open time of ative hannels. Atin filaments of various lengths were therefore tested on the Na hannel ativity by the diret use of atinbinding proteins with known speifi interations with the atin moleule. Effet of DNAse I on the atin-mediated Na hannel ativation. To further demonstrate that a "short" atin filament and not G-atin was the effetive speies responsible for the ativation of Na hannel ativity, the effet of the monomeri atin-binding protein, DNAse I, was examined in exised membrane pathes. DNAse I forms 1:1 molar omplexes with G-atin [28]. These omplexes are unable to polymerize and therefore prevent filament formation [51]. Addition of DNAse I to the ytoplasmi surfae of apial membrane pathes prior to the addition of atin prevented the stimulatory effet of exogenous atin on Na hannels, either in the presene or absene of ATP. In addition, DNAse I inhibited atin-ativated Na hannels dereasing both the average hannel number and perent open time of ative Na

5 974 'antiello: Atin regulates epithelial Na hannels 0 Q :1 8:1 16:1 32:1 No gelsolin atin:gelsolin ratio Fig. 4. Effet of gelsolin on atin -indued Na + hannel ativity. Addition of atin plus gelsolin to exised pathes with no hannel ativity indued Na hannel ativity within one minute of addition when using atin:gelsolin ratios < 8:1. Na hannel ativity is expressed as Np0, where N is the number of hannels present and p0 is the average open probability. Data at 32:1, 8:1, and 4:1 ratios are means SE of three experiments. * Statistial signifiane (P < 0.05) as ompared with data in the absene of gelsolin. Long atin filaments alone (atin polymerized for 2 hr in the presene of ATP and high ioni strength) failed to eliit Na hannel ativity (N = 4). Holding potential, 100 mv. Reprodued from [141 with permission. hannels. This effet was reversible as hannel ativity reovered with DNAse I washout but without replaing either atin or ATP to eliit this reativation, thus suggesting the ontinued presene of a pooi of short atin filaments that may be strongly attahed to the membrane [521. To further demonstrate that DNAse I binding to G-atin was the inhibiting fator on Na hannel ativity, DNAse I:atin omplexes were inubated for two hours prior to addition to the path. Addition of these omplexes to otherwise quiesent pathes was ompletely ineffetive in ativating Na hannel ativity. The enompassed evidene from the D and atin experiments strongly supports the ontention that short atin filaments were the speies responsible for Na hannel ativation. This was further explored by diretly assessing the atual length of the atin filaments involved in this ativation proess. Effet of atin.gelsolin omplexes on Na hannel ativity. The length of endogenous atin filaments is regulated by the a2- sensitive protein, gelsolin [37]. The extent to whih gelsolin will shorten atin filaments will depend on the atin:gelsolin molar ratio [37]. Thus, as an additional test of the hypothesis that atin filaments of restrited length were involved in the stimulation of Na hannel ativity, atin was polymerized in the presene of I mm a2 with different onentrations of gelsolin to eliit filaments of varying known length [37, 38]. Atin:gelsolin omplexes at a ratio of 4:1, and thus onsistent with short oligomers (tn-, tetramers) indued Na hannel ativity in exised pathes with no spontaneous ativity (Fig. 4). The ritial ratio of atin:gelsolin to eliit Na hannel ativity was < 8:1. Beause atin in the presene or absene of gelsolin was allowed to polymerize to its maximum filament length, atin alone was added as "long" filaments whih, in ontrast to the effet of short atin filaments, were ineffetive in ativating Na hannels. Effet of F-atin binding proteins on the atin-mediated Na hannel ativation. The availability of atin filaments in the viinity of the Na hannels is also an important fator in the regulatory role of atin on Na hannel ativity. Beause atin filaments are readily ross linked in the presene of suitable atin-binding proteins [22, 30], the effet of atin on Na hannel ativation was also assessed by the addition of F-atin-binding proteins to the exised pathes. Filamin, as disussed above, is known to ross link atin filaments [29, 31]. Addition of filamin to exised pathes ompletely inhibited atin-indued Na hannel ativity (Fig. 5A). The fat that spontaneous Na hannel ativity was also inhibited by the addition of filamin indiates that the endogenous atin filament organization also regulated basal hannel ativity, thus further supporting the ontention that under basal onditions, the stabilized atin filament organization may ontribute to maintain hannels in the losed state. Filamin, however, also interats with membrane glyoproteins [33], thus the possibility exists that this interation, whih is independent of filamin's ability to bind and ross link atin, ould also aount for its inhibitory effet on Na hannel ativity, This was further studied by addition of the atin-bundling protein a-atinin, known to share the same atin-binding domain as filamin but devoid of known speifi interations with plasma membrane proteins [30]. Addition of a-atinin, in ontrast to filamin, ativated Na hannels (Fig. 5B). Beause a-atinin and filamin share the same atinbinding domain [30], but only filamin is able to interat with plasma membrane glyoproteins (in ontrast to membrane lipids as is the ase of a-atinin [53]), the opposite effets of these atin-binding proteins on Na hannel ativity may be explained either by a diret interation of these proteins with speifi plasma membrane omponents inluding the Na hannel itself and/or by the alternate nature of the ross linked atin network eliited by either protein [30], whih may have differential effets on their regulatory role on Na hannels. The above findings also raise the possibility that sequenes shared by the various members of this family of atin-binding proteins an be used as tools to investigate whether speifi interations between the plasma membrane, ion hannels, and the various omponents of the atin ytoskeleton do indeed exist. This was reently explored in human melanoma ells displaying an impaired ell shape and motility whih is aused in these ells by the omplete lak of the filamin homologue ABP-280 [54]. In those and more reent studies, the suessful transfetion and expression of physiologial levels of filamin restored normal ell funtion [55]. The studies also established that the expression of filamin is required for normal ion hannel regulation in this ell model [55], sine the presene of filamin allowed a normal ell volume regulatory response after hypotoni stimulus whih was mediated by the proper regulation of volume-sensitive K hannels. Thus, atin-binding proteins may have distint effets depending on the atin onformation they enable, and thus help effet the required ell response inluding ion hannel regulation. Atin ontrols the hormonal regulation of epithelial Na+ hannels Vasopressin serves a number of roles in target tissues. In the mammalian kidney, vasoprssin modulates Na and water transport, vasoonstrition, prostaglandin prodution, and mitogeni effets. Two intraellular signaling events, either hanges in a2

6 antiello: Atin regulates epithelial Na hannels 975 ASI 2 mm after G-atin plus ATP addition Vh =60 mv 5i 4i ii 2 mm after filamin 20 nm addition :jj ::::j j:i:::i:jj:::::::j:. LA 0.5 se 21 BA ontrol V =80 mv 1 zatrr pa 0.4 se Fig. 5. Effet of filamin and a-atinin on Na + hannelativity. A. Top traing: Na hannel ativity in the presene of atin plus ATP under onditions idential to those shown in Figure 3 in an exised, inside-out path at a holding potential of 60 mv. Bottom traing: Effet of 20 nvi filamin 2 and four minutes after addition. These data are representative of 4 experiments. Reprodued with permission from [14]. B. Addition of a-atinin (10 pg/mi) to exised pathes with no spontaneous ativity indued Na hannel ativity within one minute (representative of 4, bottom traing). or AMP, are assoiated with vasopressin ation. This latter signaling pathway is assoiated with the V2-subtype of vasopressin reeptors eliiting the antidiureti hormonal effet in the kidney, perhaps the most relevant role of vasopressin in mammals. The V2-reeptor mediates the ativation of adenylyl ylase, the prodution of AMP, and the subsequent ativation of the AMP-dependent protein kinase (PIA). The phosphorylation of putative substrate targets assoiated with this physiologial response, however, have not been yet identified. The effet of vasopressin in target epithelia, whih is largely assoiated with an inrease in the apial Na permeability, is also linked to the depolymerization of atin, in a proess whih an be for the most part mimiked by ytohalasin D. Thus, in order to further understand the oupling mehanism(s) linking hormone-reeptor ativation with the physiologial response of NaF hannel ativa- tion, we also investigated the role of atin filament organization in vasopressin ation in A6 epithelial ells. In this setion, information will be summarized onerning the involvement of the AMP pathway in the A6 epithelial ell model. We will one again fous on work onduted in our laboratory pertaining to the regulatory role of the AMP-indued, and PKA-mediated ativation of the epithelial Na hannels whih are also the target of apial, pertussis toxin-sensitive G proteins [42, 48, 56] and phospholipase metabolites [561. Although several apial ion hannels are responsive to the AMP-pathway in target epithelia [57, 581, the role of the 9 ps Na hannel pool present in the apial domain of A6 ells had not been previously linked to regulation by AVP. These Na' hannels are, however, responsible for the amiloride-sensitive apial barrier to transepithelial Na4 movement in this epithelium [48].

7 976 antiello: Atin regulates epithelial Na hannels A ontrol vasopressin Vh=OmV < 1) E ) > 1.0 e -.JØ44il4* mm after 0.2 tm vasopressin ontrol Vasopressin 0.5pA se 0 ontrol Vasopressin Fig. 6. Effet 0fAVP on Na * hannelativity in A6 ells. A. Addition of AVP (0.2 rm) to ell-attahed pathes indued Na hannel ativity within five minutes as shown in this representative experiment from N = 18. Bath and pipette solutions were a 115 mi Na1 buffer as desribed in [14]. Holding potential (Vh) of zero mv refers to pipette voltage. (Right panels) AVP signifiantly inreased the average apparent number (B) and the % open probability () of the Na* hannel. *P < for AVP versus ontrol (N = 18). Reprodued with permission from [43] Effet of Al/P and the AMP pathway on Na hannel ativity To determine the role of AVP on epithelial Na hannel ativation, the effet of AVP was studied on ell-attahed pathes of partially onfluent A6 ells with methodology similar to that previously desribed for the experiments with D. Addition of AVP to ell-attahed pathes either indued or inreased Na hannel ativity within five minutes of addition (Fig. 6, for details, see [43]). The AVP-ativated hannels had a mean hannel ondutane of 8 to 10 ps, no apparent retifiation under exised, inside-out onditions, and displayed a Na :K seletivity of 3 to 5:1 as previously reported [14, 42, 46]. The data therefore indiated that AVP ativated Na hannels with similar harateristis to those previously reported in A6 ells [43, 46] and whih represent the same Na hannel pool regulated by the atin ytoskeleton [14]. The role of the AMP-mediated pathway in the regulation of the vasopressin-indued apial hannels was further explored by determining whether Na hannel ativation was also mediated by the endogenous seond messenger AMP. Three different protools were followed to inrease ytosoli AMP: (a) addition of non-hydrolyzable analogs 8-Br- or pt-amp; (b) addition of the phosphodiesterase inhibitor IBMX; and () diret ativation of adenylyl ylase with forskolin. In eah ase the addition of the drug to ells under ell-attahed path-lamp onditions resulted in a signifiant inrease in both the apparent hannel number and the average open probability, thus omparable to the results obtained with AVP (Fig. 7). Addition of the inative analog 1,9 dideoxyforskolin, however, was without effet on Na hannel ativity. Effet of PKA on Na hannel ativity The AMP-dependent pathway is responsible for the ativation of the AMP-dependent protein kinase (PKA). Therefore, to demonstrate that PKA also ativates Na hannels, exised, inside-out pathes with no apparent Na hannel ativity were first obtained. Addition of PKA and ATP to the ytoplasmi side of exised, inside-out pathes indued and/or inreased the apparent hannel number and the mean open time, thus inreasing Na hannel ativity to levels omparable to those observed by the maneuvers used to inrease intraellular AMP (Fig. 8). The effet of PKA was stritly dependent on the presene of ATP, but was independent of the presene of a2 in the bath solution. Although the AMP-ativation of apial Na hannels has been learly established for this and other related epithelia [58, 59], the steps involved in the PKA-mediated phosphorylation of substrates assoiated with Na hannel ativation in epithelia remain largely unknown. In favor of the hypothesis that regulatory proteins may

8 antiello: Atin regulates epithelial Na hannels 977 i).0 E i) i 0 i) < 1.0 a AMP IBMX Forskolin analogs Fig. 7. Effet of maneuvers to inrease AMP on Na hannel ativity in A6 ells. The data show the average apparent hannel number after addition of 8-Br-AMP or pt-amp (50 ILM; N = 6, Left), isobutyl-methylxanthine (IBMX, 100 LM; N = 3, Middle) or forskolin (50 jlm; N = 6, Right) to ell-attahed pathes with no spontaneous hannel ativity. The path-lamp onditions were as in Figure 6. Reprodued from [43] with permission. be involved in the PKA effet, data from Na hannel-ontaining vesiles have failed to show an inreased rate of Na transport in the presene of ATP and PKA as the endogenous effetors of AMP ation [60]. This is further supported by studies in whih at least one of the subunits (300 kd) of the purified epithelial Na hannel omplex from A6 ells was found to be the substrate for the speifi phosphorylation by PKA, both in vitro and in vivo [61, 62]. Although the regulatory nature of this protein on the Na hannel ativity has not been yet determined, the degree of speifi phosphorylation of this protein eliited by AVP in vivo was orrelated with an inrease in Na transport [61]. Sine ankyrin and atin have also been identified in the Na + hannel omplex [1 1], it was appealing to speulate that the atin ytoskeleton may provide a funtional link between PKA and atinassoiated regulation of Na hannels in A6 ells [14]. On the other hand, we and others have demonstrated that atin itself may be a substrate for the speifi phosphorylation by PKA [44]. Thus, despite the fat that the physiologial relevane of phosphorylated atin is largely unknown, evidene already exists for a potential role of atin phosphorylation on ell funtion. The possibility exists that phosphorylating mehanisms whih target atin may affet the native onformation of the protein sine phosphorylated G-atin loses both its ability to bind DNAse I and to undergo polymerization [51]. The effet of atin phosphorylation on polymer formation may entail an as yet unknown but relevant biologial role, sine only G- but not F-atin an be phosphorylated by protein kinase A. Thus, the pool of phosphorylated G-atin may be unable to make a ontribution to F-atin elongation. In terms of its physiologial relevane, it may be important to indiate that AVP ation has also been reently found to depolymerize atin filaments in tight epithelia [63, 64]. This event may be onsistent with our observation that short atin filaments are the only polymeri speies able to eliit Na hannel regulation [14]. Thus, the PKA-mediated phosphorylation of the atin monomer may help modulate the G-atin pool, and it is therefore tempting to postulate this mehanism as potentially relevant to the regulation of epithelial Na hannels in vivo. To investigate the role of hanges in atin filament organization in the AVP, and PKA-mediated ativation of apial epithelial Na hannels, a series of studies was onduted to determine first the role of atin on the PKA-mediated ativation of Na hannels and seond, the possible role of the speifi phosphorylation of atin on this effetor system. Exised, inside-out path-lamp experiments were thus onduted to test these hypotheses. Effet of DNAse I on the protein kinase A-ativated Na hannel ativity As indiated above, the addition of PKA in the presene of ATP was first used to indue Na hannel ativation from otherwise quiesent exised, inside-out pathes [431. Both average Na hannel number and mean open time inreased after PKA plus ATP addition. The role of the atin ytoskeleton on the PKA-ativated Na hannel ativity was next sought by addition of the monomeri (G) atin-binding protein, DNAse I. Na hannel ativity was ompletely inhibited within three minutes after addition of DNAse I (Fig. 8), an indiation that a monomeri atin pool mediated the ativation of Na hannels, and lending further support to the ontention that the ytoskeleton may be involved in the PKA-dependent ativation of Na hannels. Effet of ytohalasin D on the PKA -ativated Na hannel ativity To further assess the funtional role of the atin ytoskeleton on the PKA-mediated ativation of Na hannels, another approah was followed. As indiated above, Na hannel ativation was indued in ells exposed to D for periods of time shorter than 40 minutes. This was onsistent with the fat that a brief exposure of ells to D was devoid of marosopi hanges in ell shape [44]. Longer exposures to the drug, however, resulted in hanges in ell shape and loss of plasma membrane interations with atin. Under these onditions, Na hannel ativation was no longer observed. Membrane pathes from ells exposed to D for longer periods of time, therefore, would provide a suitable model to assess the moleular mehanisms responsible for the regulation of Na hannel ativity. Experiments were thus designed to ask the question of whether a disruption of atin filament organization alters the response to PKA in A6 ells. Exised, inside-out pathes from D-treated ells for two hours were obtained and shown to be devoid of Na hannel ativity. Interestingly, addition of PKA plus ATP to these pathes was no longer effetive in ativating Na hannels (Fig. 9). To determine that Na hannel ativity ould be resued in these ells, atin prepared as previously desribed [14] was added to the ytosoli surfae of exised pathes to form "short" atin filaments. The addition of atin under these onditions, indued Na hannel ativity within three minutes (Fig. 9). A onlusion an be drawn from these studies that atin filaments are required for the PKA-mediated ativation of apial Na hannels and support the ontention that PKA may target atin itself. This was further investigated as indiated below.

9 978 antiello: Atin regulates epithelial Na + hannels ontrol 1 mm after ATP addition Vh =60 mv. 'r 1 mm after PKA addition 8. Effet of DNAse Ion P1(4-ativated Na hannels. Addition of PIA (10 sg/ml) and ATP (1 mm) to the ytaplasmi side of exised, inside-out pathes with no spontaneous hannels (top traing), indued Na hannel 30 se after ativity within two minutes (middle traings). DNAse I addition-.fig. Subsequent addition of DNAse I (bottom traing) inhibited PKA-dependent ativation of Na hannels. The data are representative of [fa 4 experiments, and were obtained under onditions previously desribed [ se Reprodued from [44] with permission. 2i ii ontrol D, 2 hrs.a)a¼%m$$ V5 =60 mv 5 mm after PKA plus ATP addition 3 mm after 0-atmn addition $r4* LA 1 se Fig. 9. Lak of effet of P1(4 following ytohalasin D treatment. ells were treated with eytoehalasin D (D, 5 rg/ml) for at least two hours. Exised, inside-out pathes with no spontaneous Na hannel ativity were then obtained (top traing). Addition of PKA plus ATP (middle traing) did not eliit Na hannel ativation. In ontrast, subsequent addition of aetin (I mg/mi) restored Na hannel ativity within one to three minutes (bottom traing). Reprodued from [44] with permission. Atin as a substrate for phosphoiylation mediated by PIA To test the possibility that PIA ould diretly target the aetin eytoskeleton, it was hypothesized that aetin itself might be a suitable substrate for phosphorylation, and that the ovalently modified atin moleule would have properties different to those of the native aetin, The ability of PIA to phosphorylate atin in vitro was therefore determined. Aetin was shown to inreasingly phosphorylate in the presene of PIA and ATP within 60 minutes of phosphorylation. This effet was inhibited by addition of the speifi PKA inhibitor, S to 24 amide [65]. The phosphate:atin molar ratio determined under equilibrium onditions was found to be approximately two moles of phosphate per mole of aetin, and thus physiologially relevant [44]. The ability of phosphorylated atin to polymerize was next explored. The amount of F-aetin ahieved after a 15 minute period of polymerization was signifiantly lower when starting with phosphorylated atin as ompared to the native protein [44]. In ontrast, no signifiant differenes were observed in the state of polymerization between native and phosphorylated atin filaments that were first allowed to polymerize. The data demonstrate, therefore, that the PKA-mediated phosphorylation of aetin may indeed blok filament formation, but does not indue signifiant depolymerization of atin. Effet of PKA on the atin-dependent Na + hannel ativity As indiated above, phosphorylated atin has different polymerizing properties as ompared to native protein. Thus, PIAphosphorylated aetin may also have distint effets on Na

10 antiello: Atin regulates epithelial Na + hannels 979 ontrol III I.l.i. 10 mm after non-phosphorylated atin filaments addition II. washout - M4&1 ' rtir. uiri''r r'wj 1 5i 4 mm after phosphorylated atin filaments addition + 4i J I. 3i 2i ii Fig. 10. Effet of phosphoylated atin on Na hannel ativation. G-atin was phosphorylated with PKA plus ATP under non-polymerizing onditions in the presene of 600 mi potassium iodide. Non-phosphoiylated G-atin or phosphorylated G-atin was then allowed to polymerize for at least one hour. Exised, inside-out pathes with no apparent hannel ativity were obtained (top traing). Apparent hannel number inreased after addition of phosphorylated (0.2 mg/mi) (bottom traing) but not non-phosphorylated atin filaments (0.2 mg/mi) (seond traing). The data shown are representative of 2 experiments. Reprodued from 1441 with permission. hannel ativity. Monomeri atin was allowed to polymerize for at least one hour in the presene or absene of onditions suitable for phosphorylation by PKA. Atin polymerized in the presene of ative PKA (phosphorylated atin filaments) indued Na hannel ativity in exised, inside-out pathes treated with the PKA inhibitor (to prevent a diret effet of ontaminant PKA on endogenous atin, for details see [1).Atin polymerized in the absene of PKA or in the presene of inative PKA (PKA plus PKI; non-phosphoiylated atin filaments), in ontrast, did not eliit hannel ativation. This is in agreement with our previous observations that long atin filaments are without effet in ativating Na hannels [141. The stimulatory effet of phosphorylated atin filaments on Na hannels was further onfirmed with another protool to obtain phosphorylated atin. Purified G-atin was first phosphorylated under onditions in whih no polymerization ourred (detailed in [44]). Both the phosphorylated and non-phosphorylated G-atin were then allowed to polymerize as desribed above, and then tested for their ability to ativate Na hannels. The results were similar to those mentioned before. 1 pa 0.4 se Only phosphorylated atin filaments were apable of ativating Na hannels (Fig. 10). Thus, under polymerizing onditions in whih native atin filaments are unable to ativate Na hannels, phosphosylated F-atin was still apable of ativating Na hannels (Fig. 10). Two possibilities may aount for these findings. First, that phosphorylated atin filaments may have hanged their onformation in suh a way as to indue Na hannel ativation, and the seond possibility, whih is in agreement with the phosphorylation studies of atin, is onsistent with our own findings that under phosphorylating onditions, atin has a redued ability to polymerize and thus provide a long-lived pool of short atin filaments. Integration of regulatory pathways of epithelial Na hannel ativity Role of the atin ytoskeleton on the G protein-mediated and phospholipase A2-ativated Na hannels In previous studies we had demonstrated that the amilorideand AVP-sensitive apial Na hannels of A6 ells are modulated

11 980 antiello: Atin regulates epithelial Nahannels 3 hrs ontrol *r,t.4uj =80 my 5 mm after GTP7S mm after Gai-3 5 mm after arahidoni aid - -*$Mt1M [9pA 0.3 se Fig. II. Effet of atin on the G proteinmediated and phospholipase A2-ativated Na + hannel ativity. ells were treated for three hours with D (5 gg/ml) as in Figure 9. Exised inside-nut pathes with no spontaneous hannels were then obtained (top traing). Addition of either GTP7S (0.1 msi, seond traing), Gsi-3 (20 p, third traing) or arahidonie aid (10 JiM, fourth traing) were without effet on ion hannel ativity. Na hannel ativity was restored after addition of aetin (1 mg/mi) and ATP (bottom traing). Data are representative of four experiments. by another regulatory pathway involving the G protein-mediated ativation of phospholipase A2 [42, 56]. The other regulatory pathway, as indiated in this review, involves the PKA-targeted effet through atin [43, 44]. Beause both pathways are responsible for the ativation of the same apial Na hannel pool present in the plasma membrane of A6 ells, an interation between them may exist, thus linking the various regulatory steps assoiated with Na hannel ativation. The study of a funtional link between these two pathways has been therefore initiated in our laboratory with the same experimental approah used before to rid the plasma membrane of attahments to the atin filaments and thus the ativation of Na hannels, As desribed above, inubation of A6 ells with the atin filament disruptor D for three hours resulted in the ells being unresponsive to either AVP and/or PIA ativation [44]. We next evaluated the onsequenes of this maneuver on the regulatory pathway of the Na hannel ativation, involving U proteins and phospholipase A2. In ells previously treated with D, neither GTPyS nor the diret addition of the purified, ativated, U protein subunit Gaja effeted apial Na hannel ativation (Fig. 11), in ontrast to previous results in ontrol ells [42]. To further prove if ativation of phospholipase A2 was the affeted mehanism after atin filament depletion, the hydrolyti byprodut and parental substrate of the regulatory pathway, arahidoni aid, was also tested without effet. In ontrast, addition of atin plus ATP readily ativated Na hannel ativity (Fig. 11), as we have previously demonstrated in ell membranes exposed to D for two hours [44]. The data thus suggest that atin filament organization is also required to effet the proper regulatory pathway(s) involving the phospholipase A2 prodution of lipid metabolites and thus links the apial regulatory pathways of Na hannel regulation, inluding the AMP-mediated hormonal response to AVP. Thus atin is the more proximal regulatory effetor system to ontrol Na hannel ativity, therefore raising the as yet unanswered possibility that atin itself may diretly interat with the Na hannel. onlusion and perspetive The atin ytoskeleton is an essential ell omponent whose dynamis have been assoiated with a variety of ell funtions. In this ontext, hanges in atin filament organization are impliated in gel-sol transformations involved in ell movement and the ontratile properties of musle ells. More reently, the atin ytoskeleton has been also impliated in the ontrol of intraellular information assoiated with the delivery of speifi mrna [66, 67], the transfer of intraellular vesiles and reeptors [681 and the onset of hormone ation [4]. The moleular events taking plae at the plasma membrane and that modify the atin ytoskeleton are, however, ill-defined. Atin filaments are struturallyassoiated with ion transport proteins. The spetrin ytoskeleton, for example, ouples atin filaments to transmembrane proteins

12 antiello: Atin regulates epithelial Na * hannels 981 inluding the brain Na hannel, the Na,K-ATPase and the band 3 anion exhanger. The interation between atin filaments and these transport proteins has been, heretofore, impliated in the spatial distribution of these proteins to the various ell domains. ytoskeletal interations with ion transport proteins have also been funtionally impliated in the ell volume regulatory response of various ells that may involve ion hannel regulation [40, 55, 69]. ytoskeletal disruption with suh toxins as ytohalasins, for example, has been shown to obliterate the ell volume regulatory response of epithelial ells [70, 711 and other ell types [72]. The ell volume regulatory response to anisoosmoti stimuli whih is mediated by the speifi, membrane-streth ativation of the so-alled "mehano-sensitive" ion hannels [50, 73] has been thought in terms of the ativation of ion hannel ativation mediated by the strething properties of the plasma membrane. Although the moleular interations between the atin ytoskeleton and the effeted systems has not been generally provided, ion hannel ativation by mehanisms other than streth ativation have been also postulated that may diretly impliate hanges in the atin ytoskeleton instead [74]. The possibility to use the exised inside-out path lamp onfiguration in our studies provided us with a useful model for the haraterization of the moleular steps assoiating the dynamis of atin filament organization with ion hannel regulation. Our studies indiate that onformational hanges in atin are highly relevant and sine neither monomeri nor long filamental atin effets Na hannel ativation, this information would provide strong evidene for a funtional role of atin oligomers not previously available [15]. The possibility exists, however, for different ell models to respond differently to the various atin onformations. It was reently reported that the low-ondutane K hannel of the rat ortial olleting dut, otherwise sharing the same regulatory pathways as the apial Na* hannel of A6 ells [75, 76], is instead inhibited by disruption of the atin ytoskeleton with ytohalasins [771. onsidering that a variety of intraellular atin-binding proteins may extensively modify atin filament organization into its various onformations, the possibility exists for this being a novel, and perhaps, ubiquitous regulatory mehanism of ell funtion. The moleular physiology of the interation between atin and speifi ion hannels is, however, largely unknown. Atin filaments bind diretly to, and may interat with the plasma membrane and modify its strutural omposition. Reent studies, for example, indiate that phospholipase ativity an be regulated by the atin ytoskeleton [78, 791. Thus, the possibility exists for apial regulatory mehanisms other than ytoskeletal interations that may also effet epithelial Na hannel regulation, as we reently demonstrated for an apial G protein oupled to the ativation of phospholipase A2 and the prodution of arahidoni aid whih also regulates Na hannels [56]. Another, yet unexplored possibility, is the diret binding of atin to ion hannels. Although there is no information to support this ontention, new evidene has been provided by the reent loning of one epithelial Na hannel, RNa, responsible for Na reabsorption in the rat olon [80]. This hannel is atually a heterotrimer of a, /3, and y subunits, whose expression bears the funtional fingerprinting of previously reported Na hannel ativity in epithelial ells [81]. It was observed that the a subunit of RNa shares sequene homology with the atin-binding proteins known as dystrophins. The a and /3 dystrophins are impliated in the ability of musle ells to depolarize and therefore to ontrat. Interestingly, dystrophins are also members of an atinbinding family inluding spetrin, fodrin and other similar atinbinding proteins [82, 83]. Thus, the possibility exists for the arna itself to link to the atin ytoskeleton by a diret binding interation with atin. This hypothesis is supported by at least one other ion transport protein, the Na,K-ATPase, whose a subunit may bind and interat diretly with atin in suh a way as to enhane its ATP hydrolyti ativity [84]. Thus, a diret binding of atin to ion transport proteins might serve as a novel regulatory mehanism for effeting the ability of these transporters to move ions. The moleular anatomy of the interation between ion hannels and atin ytoskeleton has to be further explored. Although our studies onerned the ability of atin and its filamental strutures to modify Na hannel ativity, the possibility also exists that ion hannel ativity, as it pertains to the delivery of ions into the ytosoli ompartment, might also help modify atin filament organization and funtion. The ability of highly harged moleules suh as atin to polymerize following hanges in eletri fields has been reently postulated [85, 86]. Ion hannel ativation may thus indue a feedbak mehanism assoiated with the harge-mediated onformational hanges of atin filament organization [87]. The nature of this novel transdution mehanism has been forwarded by reent evidene indiating that atin filaments an serve as osmo-eletri transduers [87] whih may help transdue intraellular signals in the form of eletrial urrents [88]. Therefore, the atin ytoskeleton may not only help regulate ell funtion but also be the site for intraellular signaling events to take plae. Another relevant finding of our studies was the ability of atin filament organization to be modified by the speifi PKA-mediated phosphorylation of atin. Atin was diretly phosphorylated by the effetor end of a signaling mehanism triggered by AMP ativation. It is, therefore, important to onsider that regulatory mehanisms involving the onventional seond messenger pathways may effet the speifi phosphoiylation of ytoskeletal omponents assoiated with atin filaments whih in turn will help regulate ell funtion as well. In our studies, we demonstrated that AVP ativates apial epithelial Na hannels by a mehanism involving ativation of adenylyl ylase and the generation of intraellular AMP [43]. This, in turn, ativates protein kinase A. The effetor mehanism linking this interation with the ativation of epithelial Na hannels has remained illusive for almost 50 years. With the possibility to use the exised inside-out path as a probe to test the ability of protein kinase A and/or atin filaments to regulate Na hannels, we were able to unequivoally demonstrate that atin is required for the PKA effetor system to take plae (Fig. 12) [44]. Thus, AVP ation in the kidney may require an organized atin filament to eliit a physiologial response. By extension, other kinases inluding protein kinase, whih has an opposite effet on the regulation of Na hannels in epithelia, an be also postulated to target the atin ytoskeleton. Protein kinase, for example, inhibits apial epithelial Na hannel ativity in A6 ells [89]. This would be onsistent with either one of two possibilities provided that the regulation may be postulated as linked to atin filament organization. As indiated in Figure 1 and also shown by the data on the effets of DNAse I or filamin, any displaement of the pool of short atin filaments will derease the stimulatory effet of atin on the Na hannel ativity. Thus one possibility would be the atual polymerization of atin mediated

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