Biomedical subjects

G Riquelme

Publications and source records attributed to G Riquelme.

At least 19 recordsLinked to original sources

PubMed2006

Diverse calcium channel types are present in the human placental syncytiotrophoblast basal membrane.

The functional expression of calcium channels has been scarcely studied in human placental syncytiotrophoblast. We have presently sought to characterize Ca(2+) currents of the healthy syncytiotrophoblast basal membrane using purified basal membranes reconstituted in giant liposomes subjected to patch-clamp recordings. We detected presence of channels with high permeability to Ca(2+) (relative PCa/PK up to 99.5) using K(+) solutions in symmetric conditions. Recordings performed in Ba(2+) gradients showed Ba(2+)-conducting channels in 100% of experiments. Ba(2+) total patch currents were consistently blocked by addition of NiCl(2), Nifedipine (L-type voltage-gated calcium channel blocker) or Ruthenium Red (TRPV5-TRPV6 channel blocker); Nifedipine and Ruthenium Red exerted a synergic blocking effect on Ba(2+) total patch currents. Immunohistochemistry of placental villi sections evidenced presence of alpha(1) subunit of voltage-gated calcium channels and TRPV5-TRPV6 channels in basal and apical syncytiotrophoblast plasma membranes; these three calcium channels were also detected in purified basal and apical fractions using Western blot. These results show the presence of three types of calcium channels in the syncytiotrophoblast basal membrane by both functional and molecular means. These basal membrane calcium channels would not be directly involved in mother-to-fetus Ca(2+) transport, but could participate in other relevant trophoblast processes, such as exocytosis and Ca(2+) transport regulation.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗

PubMed2004

Influence of C-terminal protein domains and protein-lipid interactions on tetramerization and stability of the potassium channel KcsA.

KcsA is a prokaryotic potassium channel formed by the assembly of four identical subunits around a central aqueous pore. Although the high-resolution X-ray structure of the transmembrane portion of KcsA is known [Doyle, D. A., Morais, C. J., Pfuetzner, R. A., Kuo, A., Gulbis, J. M., Cohen, S. L., Chait, B. T., and MacKinnon, R. (1998) Science 280, 69-77], the identification of the molecular determinant(s) involved in promoting subunit tetramerization remains to be determined. Here, C-terminal deletion channel mutants, KcsA Delta125-160 and Delta120-160, as well as 1-125 KcsA obtained from chymotrypsin cleavage of full-length 1-160 KcsA, have been used to evaluate the role of the C-terminal segment on the stability and tetrameric assembly of the channel protein. We found that the lack of the cytoplasmic C-terminal domain of KcsA, and most critically the 120-124 sequence stretch, impairs tetrameric assembly of channel subunits in a heterologous E. coli expression system. Molecular modeling of KcsA predicts that, indeed, such sequence stretch provides intersubunit interaction sites by hydrogen bonding to amino acid residues in N- and C-terminal segments of adjacent subunits. However, once the KcsA tetramer is assembled, its remarkable in vitro stability to detergent or to heat-induced dissociation into subunits is not greatly influenced by whether the entire C-terminal domain continues being part of the protein. Finally and most interestingly, it is observed that, even in the absence of the C-terminal domain involved in tetramerization, reconstitution into membrane lipids promotes in vitro KcsA tetramerization very efficiently, an event which is likely mediated by allowing proper hydrophobic interactions involving intramembrane protein domains.

Amino Acid Sequence↗

PubMed2004

Isolation and purification of human placental plasma membranes from normal and pre-eclamptic pregnancies. a comparative study.

Human placental syncytiotrophoblast is the main barrier for materno-fetal exchange. Analysis of transplacental transport involves the study of ion channels in both the maternal-facing microvillous membrane (MVM) and the fetal-facing basal membrane (BM). Difficulties in having access to intact placenta with conventional electrophysiological methods favour alternative methodologies, such as isolation and reconstitution of membranes in artificial lipid systems. Pre-eclampsia is a major health problem of human pregnancy. The search for altered physiological processes in pre-eclamptic placentae requires the investigation of events at both the microvillous and basal surfaces. The aim of this study was to obtain reliable syncytiotrophoblast plasma membranes from human normal (N) and pre-eclamptic (PE) pregnancies. We describe a protocol which allows for the simultaneous isolation of MVM and BM. The purity of the membranes isolated was evaluated using enzymatic assays, binding studies, Western blotting and immunohistochemistry. Enrichment of alkaline phosphatase activity for MVM was 17 to 21-fold, with 13-16 per cent protein recovery, for both N and PE. Enrichment of adenylate cyclase activity for BM was 9-fold for N, and enrichment of dihydroalprenolol binding to beta-adrenergic receptors was 12-fold for N and 6-fold for PE, with 14 per cent protein recovery for both N and PE. Cross contamination was low and mitochondrial membrane contamination was negligible. We conclude that MVM and BM isolated from placentae of pre-eclamptic women are similar in enrichment and purity to those of healthy women, thus allowing their use in comparative electrophysiological studies.

Actins↗

PubMed2003

17beta-estradiol and tamoxifen regulate a maxi-chloride channel from human placenta.

Steroid hormones have been implicated in the modulation of several transport processes, including conductive chloride transport in epithelial cells. Micromolar concentrations of these hormones have been determined in blood of pregnant women. The purpose of this work was to explore the effects of 17beta-Estradiol, a steroid hormone, on the biophysical properties of the Maxi chloride channel present in apical membranes from human placental syncytiotrophoblast. Apical membrane chloride channels from human term placentas were reconstituted in giant liposomes suitable for electrophysiologic studies by the patch-clamp method. Low micromolar concentrations of 17beta-Estradiol inhibit the Maxi chloride channels in excised patches in a potential-dependent manner. The addition of 1 mM 17beta-Estradiol to the bath solution decreased the total current in the patch from 100% control to 71% at -40 mV holding potential and the current was not affected by 17beta-Estradiol at + 40 mV. However, the presence of the hormone did not affect the single-channel conductance, therefore its effect must be due to modulation of its open probability (Po). Interestingly, 17alpha-Estradiol did not change the total current in the patch. Tamoxifen, an antiestrogen, also showed inhibition, but in a voltage-independent manner. Our results suggest that the Maxi Cl- channel from human term placenta may be regulated by direct interaction of both compounds with the channel. From a functional point of view, the control of these channels by steroid hormones may be of great importance in placental physiology and their regulation may help to unravel their possible role in transplacental transport.

Cell Membrane↗

PubMed2002

Functional transplantation of chloride channels from the human syncytiotrophoblast microvillous membrane to Xenopus oocytes.

The materno-fetal transfer of metabolites and nutrients requires the operation of specific transport mechanisms through syncytiotrophoblast membranes. Electrophysiological studies on these cells are scarce and, because of their syncytial nature, whole-cell current recordings have not been carried out. We have now studied whether or not ion channels from the human syncytiotrophoblast microvillous (hSM) membrane can be transplanted to Xenopus oocytes. Sixty-two percent of hSM-injected oocytes displayed lower resting potential and higher membrane conductance than uninjected cells. The increased membrane conductance was due to the incorporation of Cl(-) channels, because neither replacing Na(+) in the bathing solution by N-methyl- D-glucamine or K(+), nor withdrawing Ca(2+) had any significant effect on the currents elicited by voltage pulses. In contrast, substitution of Cl(-) by different anions markedly affected the membrane conductance, giving an anion selectivity sequence of I(-)>Br(-)>Cl(-)>methanosulfonate congruent with gluconate. In addition, disulfonic stilbenes and gluconate, but not anthracene-9-carboxylic acid, blocked the transplanted channels. These properties are compatible with those of placental Cl(-) "maxi" channels. It is concluded that functional Cl(-) channels from the hSM become effectively incorporated into the Xenopus oocyte membrane, where their function can be studied in detail.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗

PubMed1999

Effect of the inactivating "ball" peptide of Shaker B on intermediate conductance Ca(2+)-dependent inwardly rectifying K+ channels of HeLa cells.

The patch-clamp technique was used to study the effect of intracellularly added inactivating "ball" peptide (BP) of the Shaker B K+ channel upon Ca(2+)-dependent inwardly rectifying K+ channels of the intermediate conductance type expressed in HeLa cells. Intracellular BP caused only moderate inhibition of outward K+ currents when assayed at an intracellular Ca2+ concentration of 100 nmol/l. Increasing intracellular Ca2+ levels led in itself to some voltage-dependent blockade of K+ currents, which was absent when high extracellular K+ was used. An additional strong blockade by intracellular BP was nevertheless observed both in Na(+)- and K(+)-rich extracellular solutions. A non-inactivating BP analogue had no effect. At this higher intracellular Ca2+ concentration the inhibition of these intermediate conductance Ca(2+)-dependent channels by BP was voltage-dependent, being absent at hyperpolarizing potentials, and could be relieved by increasing extracellular K+. These data suggest that BP acts at an internal pore site in Ca(2+)-dependent intermediate conductance K+ channels of HeLa cells, and that these might possess a receptor site for the peptide similar to that of other K+ channels such as Ca(2+)-activated maxi-K+ channels.

Calcium↗

PubMed1999

Regulation of human placental chloride channel by arachidonic acid and other cis unsaturated fatty acids.

OBJECTIVE: Arachidonic acid has been implicated in the modulation of various transport processes, including conductive chloride transport in brush border membranes in the human placenta. The purpose of this work was to explore the effects of some cis unsaturated fatty acids on the electrophysiologic properties of the maxi chloride channels present in apical membranes from human placenta. STUDY DESIGN: Apical membrane chloride channels from human term placentas were reconstituted in giant liposomes. These cell-sized liposomes, generated by a cycle of dehydration and rehydration, are suitable for electrophysiologic studies by the patch-clamp method. RESULTS: Low micromolar concentrations of arachidonic acid reversibly inhibit maxi chloride channels in excised patches. Other cis unsaturated fatty acids, such as oleic and linoleic acids, show similar blockade. The inhibition was dose dependent. The maxi chloride channel can also be inhibited by 4,4 -diisothiocyanatostilbene-2,2 -disulfonic acid, a known chloride channel inhibitor. CONCLUSIONS: Our results identify the apical membrane maxi chloride channel as a possible electrophysi ologic counterpart of 4,4 -diisothiocyanatostilbene-2, 2 -disulfonic acid and cis unsaturated fatty acid-inhibited conductance previously described in brush border membranes of the human placenta. From a functional point of view the control of these channels by arachidonic acid may be of great importance in placental physiologic characteristics. Regulation of chloride channels could be important in the control of electrolyte and fluid transfer across the placenta. In addition, if these channels contribute to setting the membrane potential their regulation could have consequences for nutrient transport and delivery to the fetus. The electrophysiologic identification of these channels and their regulation might help to unravel their possible role in transplacental transport in normal and pathologic placental tissue.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗

PubMed1998

Swelling-activated potassium currents of Ehrlich ascites tumour cells.

The K+ and Cl- currents activated by Ca2+-ionophore treatment or by hypotonic cell swelling have been studied in Ehrlich ascites tumour cells by the patch-clamp technique. A charybdotoxin-inhibitable K+ current was activated by increasing intracellular Ca2+ concentration. In contrast, the K+ current activated by cell swelling was insensitive to charybdotoxin as well as to apamin, suggesting that channels different from those sensitive to Ca2+ are responsible for regulatory volume adjustments in these cells. The magnitude of the K+ and Cl- currents activated by hypotonic challenge was markedly temperature-dependent, possibly reflecting the temperature-dependence of enzymes involved in the intracellular signalling of cell volume regulation.

Animals↗

PubMed1997

Effect of glutamate receptor phosphorylation by endogenous protein kinases on electrical activity of isolated postsynaptic densities of rat cortex and hippocampus.

Postsynaptic densities (PSDs) were isolated from rat brain cortex and hippocampus, purified and incorporated into giant (5-80 microns in diameter) liposomes. Gigaohm seals were obtained with a patch-clamp pipette, and a giant liposome PSD-containing membrane patch, was excised and recorded. The PSD was always oriented in an inside-out configuration. This allowed receptor agonists or antagonists to be added from the interior of the recording pipette, and also the addition of different substances, such as ATP, calcium, calmodulin and others to the 'intracellular' side of the PSD, i.e. to the bath. alpha-Amino-3-hydroxy-5-methylisoxazole propionic acid (AMPA) receptor agonists such as quisqualate or AMPA induced in the PSD a complex pattern of electrical activity, that was blocked by 10 microM 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), but not by 2-aminophosphonovalerate (APV). The currents generated by 0.5-1 microM quisqualate were increased by about 100% when the PSDs were phosphorylated. Similar findings were obtained when the agonist was 0.2-2 microM kainate. These currents were also blocked by a non-N-methyl-D-aspartate (NMDA) receptor antagonist but not by APV, and were increased by about 70% by phosphorylation of the PSDs. Addition of 5-10 microM NMDA plus 1 microM glycine to the 'extracellular' side of the PSD, led to a characteristic pattern of activity, with the opening of multiple receptor ion channels. This was entirely blocked by 10 microM APV. Addition of extracellular Mg2+ (1-2 mM) induced a voltage-dependent block of the currents. Phosphorylation of the PSD led to an increase of Mg(2+)-blocked current of about 80%. The effect of phosphorylation on ion channel activity showed a markedly different requirement for calcium and for calmodulin among the AMPA, kainate and NMDA types of glutamate receptors, thus suggesting that each receptor type is coupled at the synapse with a unique complement of protein phosphokinases.

2-Amino-5-phosphonovalerate↗

PubMed1997

Possible thiol group involvement in intracellular pH effect on low-conductance Ca(2+)-dependent K+ channels.

We have studied the effect of intracellular pH (pHi) shifts on the activity of Ca(2+)-dependent, inwardly rectifying K+ channels of HeLa cells. Recordings of macroscopic currents in symmetrical 145 mK K+ and internal pH of 7.4 gave moderate inward rectification of the current. At pH 6.4, inward rectification was more marked, whereas it switched to outward rectification at pH 8.2. In excised inside-out membrane patches, similar changes in pHi did not affect the single-channel conductance of the channels underlying the Ca(2+)-dependent K+ currents. At neutral pH, the open state probability (Po) was independent of voltage in the range from -70 to 70 mV. At alkaline pH, Po became voltage dependent, decreasing at negative potentials and increasing with depolarization compared with pH 7.4. These changes accounted for the pH-dependent changes in rectification of the macroscopic current. The possibility that voltage dependence might arise from the ionization of a thiol group was tested by using thiol-directed reagents. The decrease in Po produced by intracellular alkalinization at negative potential was reverted by treatment with N-ethylmaleimide, 5,5'-dithiobis(2-nitrobenzoic acid), and 2,2'-dithiodipyridine. The effect of intracellular alkalinization is speculated to occur through ionization of a cysteine group(s) within the field of the membrane affecting gating.

Calcium↗

PubMed1996

Ammonium inhibition of Ca(2+)-dependent inwardly rectifying K+ currents in HeLa cells.

The patch-clamp technique was used to examine the effect of ammonium on inwardly rectifying Ca(2+)-dependent K+ channels of HeLa cells. Ammonium added extracellularly blocked macroscopic K+ currents with an IC50 of 1.6 mM. Inhibition was fully reversible and voltage-independent and is not likely to be secondary to an increase in intracellular pH as this would have the opposite effect. In these experiments ammonium appeared to have an intracellular site of action. Intracellular ammonium rapidly and reversibly inhibits the activity of single Ca(2+)-dependent K+ channels underlying the macroscopic currents. This compound is often employed to manipulate intracellular pH but its use would not be indicated when full activity of the type of channels studied here needs to be unaltered.

Ammonium Chloride↗

PubMed1995

A chloride channel from human placenta reconstituted into giant liposomes.

OBJECTIVE: Ion channels play important roles in epithelial transport, but they are difficult to access for conventional electrophysiologic studies in intact placenta. The purpose of this work was to explore the suitability of purified trophoblast plasma membrane as a source of ion channels for reconstitution in artificial lipid membranes. STUDY DESIGN: Human placental brush border membranes were purified by differential and gradient centrifugation and fused with small liposomes. Giant liposomes were then generated by a cycle of dehydration and rehydration. These giant liposomes are suitable for electrophysiologic studies and were probed for the presence of active ion channels by the patch-clamp method. RESULTS: The results reported here indicate the presence of a high conductance chloride channel showing some similarities with "maxi" chloride channels described in secreting and absorbing epithelia. The channel had a slight outward rectification with conductances of 232 and 300 pS at negative and positive potentials, respectively. CONCLUSIONS: For the first time successful reconstitution of a human placental ion channel is achieved in a system suited for electrophysiologic studies. The chloride channel described might play a role in transplacental transport.

Alkaline Phosphatase↗

PubMed1993

Recordings of glutamate receptor channels in isolated postsynaptic densities.

We have isolated highly purified rat brain postsynaptic densities (PSDs), that are known to contain glutamate receptors of the AMPA and NMDA types. These PSDs were incorporated into liposomes, and grown, by a cycle of partial de- and rehydration in 5% ethylene glycol, into giant (5-100 microns in diameter) liposomes. These giant liposomes were then made to form Gigaohm (10-20 G omega) seals with conventional patch-clamp electrodes, which, when withdrawn, retain an excised patch in an inside-out configuration. When 5-10 microM L-glutamate (or 10 microM NMDA) plus 1 microM glycine were present inside the patch pipette, but not in the external fluid, a highly complex pattern of currents was seen in about 55% of the cases. This was characterized by very fast kinetics, conductances as high as 460 pS and multiple lower levels of 45, 80, 120, 230 and 340 pS. These currents, when evoked by NMDA plus glycine, were entirely suppressed by the NMDA antagonist 2-amino-5-phosphonovalerate, APV. However, those activated by L-glutamate plus glycine still appeared in the presence of APV in about 18% of the cases, but with lower conductance levels. Current kinetics similar to the latter ones were also induced by the AMPA receptor agonist quisqualate (10 microM) in 16% of the cases. This indicated that both NMDA and AMPA receptors were present, in a functionally well preserved state, in isolated postsynaptic densities. Indirect evidence also suggested that in our experiments, in which 212 seals were studied, only a single postsynaptic density was present in the patches in which channel activity was found.(ABSTRACT TRUNCATED AT 250 WORDS)

2-Amino-5-phosphonovalerate↗

PubMed1991

Cell to cell communication and pH in the frog lens.

Fiber cells of the lens are electrically and diffusionally interconnected through extensive gap junctions. These junctions allow fluxes of small solutes to move between inner cells and peripheral cells, where the majority of transmembrane transport takes place. We describe here a method utilizing two intracellular microelectrodes to measure the cell to cell resistance between fiber cells at any given distance into the intact lens. We also use ion-sensitive microelectrodes to record intracellular pH at various depths in the intact lens. We find that gap junctions connecting inner fiber cells differ in pH sensitivity as well as normal coupling resistance from those connecting peripheral cells. The transition occurs in a zone between 500 and 650 microns into the lens. Fiber cells peripheral to this zone have a specific coupling resistance of 1.1 omega cm2, whereas those inside have a specific coupling resistance of 2.7 omega cm2. However, when the cytoplasm of fiber cells is acidified by bubbling with CO2, peripheral cells uncouple and the cell to cell resistance goes up more than 40-fold, whereas junctions inside this zone are essentially unaffected by changes in intracellular pH. In a normal frog lens, the intracellular pH in fiber cells near the lens surface is 7.02, a value significantly alkaline to electrochemical equilibrium. Our data suggest that Na/H exchange and perhaps other Na gradient-dependent mechanisms in the peripheral cells maintain this transmembrane gradient. Deep in the lens, the fiber cell cytoplasm is significantly more acidic (pHi 6.81) due to influx of hydrogen across the inner fiber cell membranes and production of H+ by the inner fiber cells. Because of the normally acid cytoplasm of interior fiber cells, their loss of gap junctional sensitivity to pH may be essential to lens survival.

Animals↗

PubMed1990

Giant liposomes: a model system in which to obtain patch-clamp recordings of ionic channels.

Cell-size, giant liposomes have been formed by submitting a mixture of asolectin lipid vesicles and native membranes from Torpedo, highly enriched in acetylcholine receptor (AcChR), to a partial dehydration/rehydration cycle [Criado, M., & Keller, B. U. (1987) FEBS Lett. 224, 172-176]. Giant liposomes can be prepared in bulk quantities, in the absence of potentially damaging detergents or organic solvents, and their formation is mediated by membrane fusion phenomena. In fact, fluorescence microscopy and freeze-fracture data indicate that protein and lipid components of the initial membranes and lipid vesicles are homogenously distributed in the resulting liposomes. Giant liposomes containing AcChR have been used as a model to evaluate whether this system can be used to monitor the activity of ionic channels by using high-resolution, patch-clamp techniques. Excised liposome patches in an "inside-out" configuration have been used in this work. We find that the most frequent pattern of electrical activity in response to the presence of acetylcholine in the patch pipet corresponds to a cation-specific channel exhibiting a dominant conductance level and a sublevel of approximately 78 and 25 pS, respectively. Such channel activity exhibits the pharmacological specificity, ion channel activation, ion selectivity, and desensitization properties expected from native Torpedo AcChR. Thus, it appears that the giant liposome technique offers a distinct advantage over other reconstitution procedures in that it provides a unique opportunity to undertake simultaneous biochemical, morphological, and electrophysiological studies of the incorporated ionic channel proteins.

Animals↗

PubMed1990

Agonist binding to purified glycine receptor reconstituted into giant liposomes elicits two types of chloride currents.

Using 'inside-out' membrane patches obtained from reconstituted giant liposomes containing purified glycine receptor from rat spinal cord, we have detected chloride currents elicited in response to the presence of the agonists glycine or beta-alanine. Regardless of the agonist employed, two different patterns of single channel currents could be detected, which differ in their main conductance, complexity of substates and opening frequency. In agreement with the expectations of glycine receptor heterogeneity suggested recently at the mRNA and cDNA level, our results indicate the existence of functionally different glycine receptors in the adult rat spinal cord.

Alanine↗

PubMed1986

Transverse tubules from frog skeletal muscle. Purification and properties of vesicles sealed with the inside-out orientation.

Transverse tubule vesicles were isolated from frog skeletal muscle by a procedure initially described by Rosemblatt et al. (J. Biol. Chem. 256, 8140-8148 (1981)) and later modified by Hidalgo et al. (J. Biol. Chem. 258, 13937-13945 (1983]. A large fraction of the isolated vesicles (80-90%) were sealed, as indicated by the detergent induced increase in (Na+ + K+)-ATPase activity and ATP-dependent ouabain binding. To determine the orientation of the sealed vesicles binding of digoxin, a lipid soluble derivative of ouabain, was measured. The same values of ATP-dependent digoxin binding were found with or without detergents, indicating that all the vesicles that are sealed have the ATP site accessible, and hence are sealed with the cytoplasmic side-out (inside-out orientation). The transverse tubule preparation isolated from frog muscle is highly purified, as indicated by its cholesterol content and its (Na+ + K+)-ATPase activity; negligible contamination with sarcoplasmic reticulum was observed, as indicated by the protein composition and the lack of measurable Ca2+-ATPase activity of the isolated transverse tubules. High initial rates of Mg2+-ATPase activity were found, with the peculiar property of being inhibited during the course of the reaction. Addition of lysophosphatidylcholine or saponin partially prevented the inhibition of Mg2+-ATPase activity during the reaction.

Animals↗

PubMed1982

Lipid monolayer expansion by calcium-chlorotetracycline at the air/water interface and, as inferred from cell shape changes, in the human erythrocyte membrane.

Chemically induced shape changes of the human erythrocyte may result from cell membrane bending by surface tension changes at the lipid bilayer (Evans. E.A. (1974( Biophys. J. 14, 923-931) implicating differential expansion of the monolayers coupled to form the red cell membrane (Sheetz, M.P. and Singer, S.J. (1974) Proc. Natl. Acad. Sci. U.S.A. 71, 4457-4461). Interacting with calcium, the antibiotic chlorotetracycline (CTC) transforms crenated cells (echinocytes) into cup-shaped ones (stomatocytes), presumably expanding thereby the red cell membrane inner leaflet relative to the outer one (Behn, C., Lübbemeier, A. and Weskamp, P. (1977) Pflügers Arch. 372, 259-268). Whether the Ca-CTC interaction with lipid monolayers may in fact expand the latter, has now been examined by surface tension measurements at the air/water interface. CTC and lipids appeared to compete for the available sites at the air/water interface, contributing additively to its surface pressure. Ca increased both the adsorption rate of the antibiotic to the interface and the CTC-induced surface pressure increment. The latter was not influenced by the subphase pH and ionic strength, or by the type of phospholipid polar head. Correspondingly, CTC-induced cell shape changes should be determined by the pCa values facing either monolayer of the erythrocyte membrane. Both stomatocytes and echinocytes could indeed by obtained with 0.5 mmol . 1(-1) CTC, the cell shape depending on whether the external medium was adjusted respectively to pCa 9 or to pCa 3. Fluorescence microscopy revealed the Ca-CTC complex to be mostly restricted to the cell in stomatocytes and to the external medium in echinocytes. The possibility of inducing alternative cell shapes by varying the transmembrane Ca-CTC distribution, and the demonstration of a Ca-dependent expansion of even relatively compressed lipid monolayers by CTC, together suggest that the Ca-CTC complex may also differentially expand either leaflet of the red cell membrane.

Air↗