Annexin 6 Modulates the Maxi-chloride Channel of the Apical Membrane of Syncytiotrophoblast Isolated from Human Placenta

The syncytiotrophoblast separates the maternal and fetal blood and constitutes the primary barrier for maternal-fetal transport. The Maxi-chloride channel from the apical membrane of the syncytiotrophoblast plays a role in the chloride conductance. Annexins can play an important role in the regulation of membrane events. In this study we evaluate the role of annexin 6 in the Maxichloride channel properties. The results showed that annexin 6 is bound in the apical placenta membranes in a calcium-dependent phospholipid-binding manner but also in a calcium-independent fashion. The neutralization of annexin 6 decreased the total current by 39 ± 1.9% in the range of ±80 mV, and the currents decrease with the time. The single-channel slope conductance was decreased from 253 ± 7.4 pS (control) to 105 ± 13 pS, and the amplitude decreased by 50%. The open probability was also affected when higher voltage steps were used, changes in either the positive or negative direction induced the channel to close, and the open probability (Po) did not decrease. In channels with neutralized annexin 6, it was maintained at 1 at ±40 mV and at ±80 mV. These results suggest that endogenous annexin 6 could regulate the Maxi-chloride channel. The results obtained with normal placentae, in which annexin 6 was neutralized, are similar to those described for the Maxi-chloride channel isolated from pre-eclamptic placenta. Together these data suggest that annexin 6 could play an important role in ion transport of the placenta. The syncytiotrophoblast separates the maternal and fetal blood and constitutes the primary barrier for maternal-fetal transport. The Maxi-chloride channel from the apical membrane of the syncytiotrophoblast plays a role in the chloride conductance. Annexins can play an important role in the regulation of membrane events. In this study we evaluate the role of annexin 6 in the Maxichloride channel properties. The results showed that annexin 6 is bound in the apical placenta membranes in a calcium-dependent phospholipid-binding manner but also in a calcium-independent fashion. The neutralization of annexin 6 decreased the total current by 39 ± 1.9% in the range of ±80 mV, and the currents decrease with the time. The single-channel slope conductance was decreased from 253 ± 7.4 pS (control) to 105 ± 13 pS, and the amplitude decreased by 50%. The open probability was also affected when higher voltage steps were used, changes in either the positive or negative direction induced the channel to close, and the open probability (Po) did not decrease. In channels with neutralized annexin 6, it was maintained at 1 at ±40 mV and at ±80 mV. These results suggest that endogenous annexin 6 could regulate the Maxi-chloride channel. The results obtained with normal placentae, in which annexin 6 was neutralized, are similar to those described for the Maxi-chloride channel isolated from pre-eclamptic placenta. Together these data suggest that annexin 6 could play an important role in ion transport of the placenta. The human placental syncytiotrophoblast is a polarized epithelial structure that results from the fusion of precursor cytotrophoblast cells producing a syncytium. It is the main barrier for materno-fetal exchange. Ionic transport in the syncytiotrophoblast involves conductive pathways that are associated with numerous epithelial functions, such as maintenance of membrane voltage, cell volume regulation, solute transport, and others. In placenta the 4,4′diisothiocyanostilbene-2,2′-disulphonic acid (DIDS)-sensitive Cl- conductances and K+ conductances contribute to the resting potential of the syncytiotrophoblast microvillous membrane and are involved in volume regulation (1Birdsey T.J. Boyd R.D. Sibley C.P. Greenwood S.L. Am. J. Physiol. 1999; 276: R1479-R1488PubMed Google Scholar, 2Clarson L.H. Greenwood S.L. Mylona P. Sibley C.P. Placenta. 2001; 22: 328-336Crossref PubMed Scopus (23) Google Scholar). Chloride is the main anion of extracellular fluid in the fetus, as it is in the adult, but at all gestational ages fetal Cl- is 5–6 mm higher than in maternal blood. There are no maternal-fetal differences in either Na+ or K+ concentration (3Bissonnette J.M. Weiner C.P. Power Jr., G.G. Placenta. 1994; 15: 445-446Crossref PubMed Scopus (11) Google Scholar). There has been considerable interest focused on the chloride conductance of the human placenta apical membrane and how it is regulated. However, the specific ion channels involved in these and other placental processes are as yet unknown. A possible molecular candidate for the 4,4′diisothiocyanostilbene-2,2′-disulphonic acid-sensitive anion conductance in apical syncytiotrophoblast plasma membrane is a Maxi-chloride channel. Maxi-chloride channels have been identified in secreting and absorbing epithelia (4Halm D.R. Frizzell R.A. J. Gen. Physiol. 1992; 99: 339-366Crossref PubMed Scopus (85) Google Scholar), in non-epithelial cell types (5Blatz A.L. Magleby K.L. Biophys. J. 1983; 43: 237-241Abstract Full Text PDF PubMed Scopus (191) Google Scholar, 6Gray H.H. Johnson V.E. Poston L. Hilton P.J. J. Hypertens. Suppl. 1984; 2: S467-S469Crossref PubMed Scopus (13) Google Scholar, 7Nelson D.J. Tang J.M. Palmer L.G. J. Membr. Biol. 1984; 80: 81-89Crossref PubMed Scopus (121) Google Scholar, 8Nobile M. Galietta L.J. Biochem. Biophys. Res. Commun. 1988; 154: 719-726Crossref PubMed Scopus (32) Google Scholar), and in the apical membrane from human placenta using electrophysiological methods by our group and by others (9Brown P.D. Greenwood S.L. Robinson J. Boyd R.D. Placenta. 1993; 14: 103-115Crossref PubMed Scopus (46) Google Scholar, 10Riquelme G. Parra M. Am. J. Obstet. Gynecol. 1999; 180: 469-475Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar, 11Riquelme G. Stutzin A. Barros L.F. Liberona J.L. Am. J. Obstet. Gynecol. 1995; 173: 733-738Abstract Full Text PDF PubMed Scopus (31) Google Scholar). This channel may play a role in complex cellular regulation involving inhibition through phosphorylation by protein kinase C, activation by a Ca2+-dependent process, and in volume regulation. More recently, Sabirov et al. (12Sabirov R.Z. Dutta A.K. Okada Y. J. Gen. Physiol. 2001; 118: 251-266Crossref PubMed Scopus (186) Google Scholar) concluded that this channel serves as a pathway for swelling-induced ATP release. In normal human term placentae, the Maxi-chloride channel from the apical membrane of the syncytiotrophoblast has similar biophysical characteristics to the other Maxi-chloride channels in epithelial and non-epithelial cells. There is evidence that suggests that the cytoskeleton participates in the regulation of the Maxi-chloride channels involved in volume regulation decrease. In the non-swelling rabbit cortical collecting cell line RCCT-28A, agents that depolymerize F-actin enhance the chloride current by activation of the Maxi-chloride channel. On the other hand, F-actin-stabilizing agents prevent the activation of this channel. This evidence suggests that desegregation of the cytoskeleton activates Maxi-chloride channels and could be the reason why the activity of the Maxi-chloride channel is detected mainly in isolated patches. In 1999 Babiychuk and collaborators (13Babiychuk E.B. Palstra R.J. Schaller J. Kampfer U. Draeger A. J. Biol. Chem. 1999; 274: 35191-35195Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar) found that annexin 6 participated in the formation of reversible membrane cytoskeleton complexes in smooth muscle cells. Annexins are membrane organizers that interact with actin and could be determining factors in complex formation between the cytoskeleton and the plasma membrane. Annexins are a family of calcium-dependent phospholipid-binding proteins. Calcium reversibly shifts the annexins from a soluble to a membrane-associated state (14Crumpton M.J. Dedman J.R. Nature. 1990; 345: 212Crossref PubMed Scopus (193) Google Scholar, 15Seaton B.A. Dedman J.R. Biometals. 1998; 11: 399-404Crossref PubMed Scopus (54) Google Scholar). Several reports show that specific members of the annexin family participate in the regulation of ionic channels in different cells (16Chan H.C. Kaetzel M.A. Gotter A.L. Dedman J.R. Nelson D.J. J. Biol. Chem. 1994; 269: 32464-32468Abstract Full Text PDF PubMed Google Scholar, 17Diaz-Munoz M. Hamilton S.L. Kaetzel M.A. Hazarika P. Dedman J.R. J. Biol. Chem. 1990; 265: 15894-15899PubMed Google Scholar, 18Naciff J.M. Kaetzel M.A. Behbehani M.M. Dedman J.R. J. Comp. Neurol. 1996; 368: 356-370Crossref PubMed Scopus (46) Google Scholar, 19Nilius B. Gerke V. Prenen J. Szucs G. Heinke S. Weber K. Droogmans G. J. Biol. Chem. 1996; 271: 30631-30636Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). Naciff et al. (20Naciff J.M. Behbehani M.M. Kaetzel M.A. Dedman J.R. Am. J. Physiol. 1996; 271: C2004-C2015Crossref PubMed Google Scholar) showed that annexin 6 regulates the potassium and calcium currents of sensorial neurons. By using nonspecific affinity-purified anti-annexin 6 antibody they found an increase in the magnitude of the K+ current and an increase in the Ca2+ current in sensory neurons. These reports suggest that endogenous annexin 6 regulates the Ca2+ conductance, which indirectly modifies Ca2+-dependent ionic conductances in neurons. Diaz-Muñoz et al. (17Diaz-Munoz M. Hamilton S.L. Kaetzel M.A. Hazarika P. Dedman J.R. J. Biol. Chem. 1990; 265: 15894-15899PubMed Google Scholar) demonstrated that annexin 6 increased the mean open time and opening frequency of the sarcoplasmic reticulum ryanodine-sensitive calcium channels. In placenta, annexin 6 is expressed in cytotrophoblasts, syncytiotrophoblasts, fetal vascular endothelium, and chorionic trophoblasts throughout pregnancy (21Rambotti M.G. Spreca A. Donato R. Cell Mol. Biol. Res. 1993; 39: 579-588PubMed Google Scholar). The aim of the present study was to evaluate the role of annexin 6 in the regulation of the Maxi-chloride channel of apical membranes isolated from placentae from normal subjects and reconstituted in giant liposomes. The results showed that the electrophysiological characteristics of chloride channels with neutralized annexin 6 are different from those with intact annexin 6. These results suggest that annexin 6 has the ability to regulate the Cl- conductance in human placenta. Placenta Collection—Placentae obtained from normal pregnancies were collected immediately after delivery from the San José Hospital Maternity Unit and transported to the laboratory on ice. Immunohistochemistry of and placental were placentae were at annexin 6, we the for the antibody annexin 6 with which a as the were with for 1 and in was as a negative were by was to and and of the were of human placental microvillous membrane microvillous enhance anion pS, were by a we have described V. M. P. G. Placenta. PubMed Scopus Google Scholar) that of apical and membranes from the placenta. This is a of the described by et al. A. M.M. Biophys. 1990; PubMed Scopus Google Scholar). to plasma membrane of membranes R. P. Biochem. 1993; PubMed Scopus Google Scholar) and have the in our apical membrane when with this The involved of a membrane with and a were with mm A of the of protein was on the The at the was collected and with mm at for The was in mm mm and at The and of the membrane was using and for membrane of activity apical membrane for was to with protein to the and was of membranes and of of apical membranes from and membranes was by of activity and of to membrane and as from the placenta were in and and at for were and using at mV. molecular were as The were membranes The were for 1 in of mm mm and at with The were with of anti-annexin 6 antibody at The were in for 1 at with rabbit at and with was with using an of the were by a of the isolated apical membrane and to a as by et al. G. A. J.M. Jr., 1990; 276: PubMed Scopus Google Scholar). of membrane protein was with of a 13 mm of of the the the of the giant from to of of giant were the and with of the of for were obtained by as described by et al. A. B. PubMed Scopus Google Scholar). were on giant with of to of the from the in an was with an at a of and a of The potential was to the of the and the was maintained at The was an and the potential was for when The was by of the of single-channel and were at and the calcium and are expressed as mean ± of were obtained using A of than was of 6 in results show that of placental from normal subjects at term for annexin 6 in the apical and syncytiotrophoblast membranes The of from to also in the fetal vascular 6 in from that annexin 6 is present in the in the electrophysiological in the of the calcium and in the of we using a specific annexin 6 can be in all the annexin 6 is in the These data suggest that the normal of the annexin 6 is bound to the membrane and it is not the evaluate the of annexin 6 in the membranes were on in the of a calcium were and and annexin 6 were by by in the and in the The results showed that the with of the annexin 6 the which also suggests that is a of annexin 6 that the membranes in a calcium-independent fashion. all the annexin 6 it was to a It is that by from the membrane. apical membranes were isolated in the of mm throughout the they annexin 6 than those isolated in the of the A and These results that annexin 6 is bound in the placenta membranes in a calcium-dependent manner but also in a calcium-independent fashion. 6 the Maxi-chloride from that annexin 6 is present in a possible role for annexin 6 in of the Maxi-chloride channel present in these this we the of anti-annexin 6 antibody on the activity of the Maxi-chloride channel by the antibody to the in of the Maxi-chloride channels. The Maxi-chloride channel was to and the of the to the The of the by annexin 6 antibody was by it to the inhibition by the The were in Cl- was the as with the current to and to either or annexin 6 The results the results in which we showed a activity of the conductance of the chloride channels with different for the Maxi-chloride channel from channel current at the for chloride channels are in The in of normal Maxi-chloride and in and show the for the chloride currents with and with annexin 6 antibody in the at show types of activity for the Maxi-chloride channel in these The is similar to the that did not the activity in the However, when annexin 6 is neutralized by was a in the biophysical of the channel. the biophysical of the affected conductance, the channel open probability (Po) was with neutralized and annexin 6 of 6 on in the of annexin 6 antibody to the decreased the total current in the by 39 ± 1.9% in the range of ± mV However, the of did not the total current in the show the results at the different in The time of total current in the that the current did not with time either or in the of However, when the annexin 6 antibody was to the the currents decreased with time on total current of the Maxi-chloride in the of and C, in the annexin 6 to the at the for Maxi-chloride from was obtained with at mV for the the or for annexin 6 the current was used, for in These conductances were in with the and and M. G. J. Membr. Biol. PubMed Scopus Google Scholar, 10Riquelme G. Parra M. Am. J. Obstet. Gynecol. 1999; 180: 469-475Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar, 11Riquelme G. Stutzin A. Barros L.F. Liberona J.L. Am. J. Obstet. Gynecol. 1995; 173: 733-738Abstract Full Text PDF PubMed Scopus (31) Google Scholar) the single-channel slope conductance was 253 ± 7.4 pS for the Maxi-chloride channel in The activity in the of in the did not show changes in conductance ± the single-channel slope conductance in the of annexin 6 antibody decreased to 105 ± 13 pS The single-channel amplitude was affected by annexin 6 with a decrease of (Po) for Maxi-chloride from voltage of open channel probability of apical chloride channels has been obtained as the current of current to a voltage in These were as is the is the voltage, and is the conductance mV. A voltage was from mV to mV, at a of the for voltage for these In with the characteristics of for the placental Maxi-chloride channel from normal placenta (9Brown P.D. Greenwood S.L. Robinson J. Boyd R.D. Placenta. 1993; 14: 103-115Crossref PubMed Scopus (46) Google Scholar, M. G. J. Membr. Biol. PubMed Scopus Google Scholar, 10Riquelme G. Parra M. Am. J. Obstet. Gynecol. 1999; 180: 469-475Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar) the open probability (Po) voltage for our normal could be described by a The channel was open at between and mV, and voltage in either a positive or negative direction induced channel A similar was in the of in the In the of annexin 6 antibody the channel was also open at between mV and at higher voltage in either a positive or negative no channel were did not it is maintained at 1 at ±40 mV and 1 at ±80 mV in channels with neutralized annexin 6, with that from at ±40 mV to at ±80 mV in the in the of voltage of for neutralized annexin 6 is with to that of channels with neutralized annexin 6 a voltage of The results in this study data by other that showed that annexin 6 is expressed in the syncytiotrophoblast of the placenta. However, in to other we that annexin 6 is in the apical and membrane of the placenta The of annexin 6 at the plasma membrane suggests a potential role in the plasma membrane we have identified Maxi-chloride channels in the apical membrane of the human placentae, we to evaluate annexin 6, which is expressed in the apical regulates the electrophysiological of the Maxi-chloride channel. was to a anti-annexin 6 antibody to the annexin 6 protein bound to the apical membrane The of specific to of annexin family members has been (16Chan H.C. Kaetzel M.A. Gotter A.L. Dedman J.R. Nelson D.J. J. Biol. Chem. 1994; 269: 32464-32468Abstract Full Text PDF PubMed Google Scholar, 17Diaz-Munoz M. Hamilton S.L. Kaetzel M.A. Hazarika P. Dedman J.R. J. Biol. Chem. 1990; 265: 15894-15899PubMed Google Scholar, J.M. Behbehani M.M. Kaetzel M.A. Dedman J.R. Am. J. Physiol. 1996; 271: C2004-C2015Crossref PubMed Google Scholar). apical membrane giant to evaluate the role of this protein on the regulation of the Maxi-chloride channel. of the protein was affected by the for apical and placental results showed that the apical membrane of annexin 6. However, when we to an apical membrane in the of annexins by to the P. Biophys. 1994; PubMed Scopus Google Scholar, 15Seaton B.A. Dedman J.R. Biometals. 1998; 11: 399-404Crossref PubMed Scopus (54) Google Scholar, M.A. B.A. Biophys. 1994; PubMed Scopus Google Scholar), we calcium-dependent and calcium-independent of annexin 6. the of annexin 6 were between in the and in the of we an of annexin 6 that of the annexin 6 is bound to the apical membrane in a calcium-independent fashion. This with results by and collaborators showed that changes in the and concentration of the with the annexin 6 and P. J. U. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). 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PubMed Scopus Google Scholar). evaluate annexin 6 was associated with or bound to a that was to our membrane is a of in that a when in a that this group of A of have been to and on the muscle S. et of Scholar). In our when the was to the membrane it induced of the annexin 6 from the which of annexin 6 to we the of annexin 6 in the apical we the of neutralization of this protein on the Maxi-chloride channel we the characteristics of the channel in the of antibody and in the of the specific to the of a nonspecific to of the The results showed no between the and the with the However, when the anti-annexin 6 antibody was to the a was in the of the Maxi-chloride channel the annexin 6 antibody also decreased the conductance of the channel and affected the open probability of the channel. These results suggest that the between annexin 6 and the annexin 6 antibody may the of annexin 6 to the or may the between the protein and the Maxi-chloride channel. the of the antibody is specific to annexin 6 and the did not have we that this is to annexin 6 and not to data also suggest that annexin 6 is a of the Maxi-chloride channel. placental transport is for the of the fetus, these results are of and may open for the study of the role of annexin 6 and regulation, in such as and results from laboratory L. P. G. Placenta. PubMed Scopus Google Scholar) have a decrease in the conductance a decrease in the open and a when the Maxi-chloride channel was in membrane isolated from with similar to the the annexin 6 was These results suggest that annexin 6 may play a role in the transport of normal and The that annexin 6 was found to at also suggests that the and of annexin 6 may have an in it is that in the is a decrease in M. A. J. R. A. S. J. 2001; 15: Google Scholar, M. A. J. 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Full Text Full Text PDF PubMed Scopus Google Scholar) have that a in has a structure similar to those of channels and that human of are channels. was found to be in a channel from human the channel showed current voltage, acid However, the of the channel did not show a an channel. In this the showed that the family or a conductance Cl- channel. In the with the of in human that it was found in the and blood other but not in placenta. In the it be to a to the that the channel from human placenta to annexin 6 with the channel. that annexin 6 with the Maxi-chloride channel in human be using the anti-annexin 6 antibody and channel antibody in a similar as has been in other to show the between the J. U. S. A. 99: PubMed Scopus Google Scholar). The results suggest that annexin 6 may be a of placental transport and that this is on the state of the syncytiotrophoblast cells. in the can the of annexin 6 on the Maxi-chloride channel. This can be of the changes found in pregnancy with are to evaluate the role of the annexin 6 in are to M. and the at The San José Hospital Maternity to for with the and for of this also M. and for to the

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