Effects of palytoxin on cation occlusion and phosphorylation of the (Na+,K+)-ATPase.

Palytoxin (PTX) inhibits the (Na(+) + K+)-driven pump and simultaneously opens channels that are equally permeable to Na+ and K+ in red cells and other cell membranes. In an effort to understand the mechanism by which PTX induces these fluxes, we have studied the effects of PTX on: 1) K+ and Na+ occlusion by the pump protein; 2) phosphorylation and dephosphorylation of the enzyme when a phosphoenzyme is formed from ATP and from P(i); and 3) p-nitro phenyl phosphatase (p-NPPase) activity associated with the (Na+, K+)-ATPase. We have found that palytoxin 1) increases the rate of deocclusion of K+(Rb+) in a time- and concentration-dependent manner, whereas Na+ occluded in the presence of oligomycin is unaffected by the toxin; 2) makes phosphorylation from P(i) insensitive to K+, and 3) stimulates the p-NPPase activity. The results are consistent with the notion that PTX produces a conformation of the Na+, K(+)-pump that resembles the one observed when ATP is bound to its low-affinity binding site. Further, they suggest that the channels that are formed by PTX might arise as a consequence of a perturbation in the ATPase structure, leading to the loss of control of the outside "gate" of the enzyme and hence to an uncoupling of the ion transport from the catalytic function of the ATPase.

Ion channels formed by transcription factors recognize consensus DNA sequences.

Transcription factors (TFs) are proteins which bind to specific DNA sequences and thus participate in the regulation of the initiation of transcription. We report in this communication our observations that several of these proteins interact with lipid membranes and form ion-permeable channels. For each of the TFs that we studied, the single channel conductance was distinctively different, i.e. each TF had its own electrical signature. More importantly, we show for the first time that addition of cognate double-stranded DNA sequences leads to a specific response: an increase in the conductance of the TF-containing membrane. Strikingly, the effect of cognate DNA was observed when it was added to the trans-side of the membrane (opposite to where the TF was added), strongly suggesting that the TFs span the membrane and that the DNA-binding domain is trans-accessible. Alterations in the primary structure of the TF factors in their basic and DNA-binding regions change the characteristics of the conductance of the protein-containing membranes as well as the response to DNA addition, reinforcing the notion that the changes we measure are due to specific interactions.

How one teaching hospital system and one medical school are jointly affirming their academic mission.

The economic forces that are reshaping the practice of medicine and the funding of medical research will have great impact on clinical education and research in teaching hospitals and their associated medical schools. Changes in the setting of and approach to medical education will need to be made in order to continue to train physicians at the same high level as in the past and to maintain the productivity of our national biomedical research enterprise and its contributions to health. Academic leaders, such as department chiefs who have clinical service responsibilities, are finding it more and more difficult to manage simultaneously the demands of the clinical business, education, and research. In an effort to organize a teaching hospital and a medical school in a manner that would position them to maintain more effectively their common academic mission front and center with the clinical business, Harvard Medical School and the Beth Israel Hospital created a joint venture in 1996. The new nonprofit Institute for Education and Research has education and research as its top (and only) mission. It is designed to provide additional and specific academic leadership and to enable the joint venture to undertake strategic planning for the academic mission. In addition to the challenges it faces from changes in the external environment, the Institute for Education and Research will need to establish a new pattern of interactions internally within the parent institutions. Collaborations with department chairs and faculty are an essential ingredient for its success. It is hoped that this structure will prove to be a useful template for organizing other medical school-hospital collaborations on behalf of the academic mission.

Interaction of palytoxin with red cells: structure-function studies.

Palytoxin (PTX), a potent toxin isolated from the marine soft coral Palythoa tuberculosa increases the cationic permeability of red cell membranes and inhibits the (Na+,K+)-activated ATPase, effects that are completely reversed by ouabain. It has also been shown to compete with ouabain for a binding site and it has been suggested that it binds to the Na+,K(+)-pump molecule in cells. In a search for analogues of PTX that would bind covalently and could thus be used to identify and characterize the binding site, we have used compounds which differed from PTX at one end or at both ends simultaneously. In order to determine whether these derivatives could be used to replace palytoxin, we tested their potency to induce an increased cation flux, complete with ouabain for its binding site, and inhibit the isolated, purified (Na+,K(+)-ATPase). The results obtained indicate that departures from the PTX structure at one end or at both ends simultaneously substantially decrease the ability of the compounds to increase the cationic permeability of red blood cells and to inhibit the (Na+,K(+)-ATPase). These actions were found to be completely reversed by ouabain, but the analogues are two to three orders of magnitude less potent than PTX in competing with ouabain for its binding site. These results suggest that both ends of the palytoxin molecule participate in the interactions of the toxin with its receptor and that modifications in these regions of the molecule produce significant alterations in its binding and subsequent action on red cell membranes.

Inhibition of the actions of palytoxin on the K+ efflux from red cells and on the Na/K-ATPase activity by a monoclonal antibody.

Palytoxin (PTX) binds to the Na/K pump, inhibits the (Na/K)-ATPase and forms Na and K permeable channels in human red cells. Here, we report that a monoclonal antibody raised against a derivative of PTX (Bignami, G.S. et al. (1992) Toxicon 30, 687-700) inhibits these effects. The observations are consistent with a model in which (a) the antibody binds and, thus reduces the concentration of free PTX available to react with the Na/K pump, and, (b) the PTX-antibody complex also binds to the PTX receptor on the Na/K pump but in such a way that a cation permeable channel is not formed, probably by reducing the concentration of free PTX. Using this model, we estimate that the apparent dissociation constant for the binding of PTX to antibody is 0.2 nM.

Palytoxin induces an increase in the cation conductance of red cells.

Palytoxin (PTX), isolated from the marine soft coral Palythoa tuberculosa, increases the cation conductance of human red cell membranes. In the presence of 10(-10) M PTX and 10(-5) M DIDS, the membrane potential approximates the equilibrium potential for Na+ or K+ rather than Cl-. Even in the absence of DIDS, the Na+ and K+ conductances were greater than the Cl- conductance. The selectivity of the PTX-induced cation conductance is K+ greater than Rb+ greater than Cs+ greater than Na+ greater than Li+ much greater than choline+ greater than TEA+ much greater than Mg2+. Measurements of K+ efflux revealed two apparent sites for activation by PTX, one with a Kal of 0.05 nM and a maximum flux, nu max1, of 1.4 mol/liter of cells per h and another with a Ka2 of 98 nM and a nu max2 of 24 mol/liter of cells per h. These effects of PTX are completely blocked by external ouabain (300 microM) and prevented by internal vanadate (100 microM). When the PTX channels are open, the Na,K pumps do not catalyze ATP hydrolysis. Upon thorough washout of cells exposed to about five molecules of PTX/pump, the Na,K pump of these cells operates normally. Blockage of the positively charged NH2 terminus of PTX with a p-bromobenzoyl group reduces the potency of the compound to induce Na and K fluxes by at least a factor of 100, and to compete with the binding of [3H]ouabain by at least a factor of 10. These data are consistent with the conclusion that PTX binds reversibly to the Na,K pumps in the red cell membrane and opens a (10-pS) channel equally permeable to Na and K at or near each pump site.

CD4+ lipid bilayers. A model for human immunodeficiency virus type 1 coat protein binding.

gp120, the coat glycoprotein of the human immunodeficiency virus type 1 (HIV1) binds to a molecule on the surface of a class of T-lymphocytes, CD4, which is also the receptor for major histocompatibility complex class II (MHCII). To study the events that follow the interaction of gp120 with CD4, we have incorporated CD4 into lipid bilayers and recorded the electrical changes which occur after the addition of gp120. Interaction of gp120 to CD4-containing bilayers induces multistate ion-permeable channels with a maximum conductance of 380-400 picosiemens. When CD4+ bilayers were preexposed to either MHCII or to OKT4A antibody, no channels were formed after the addition of gp120. These results indicate that CD(4+)-containing bilayers bind gp120, MHCII, and OKT4A, that binding of gp120 produces ion-permeable channels, and that CD4+ bilayers can be used to assay for gp120 in the solution bathing the bilayer.

Primary structure of peptides and ion channels. Role of amino acid side chains in voltage gating of melittin channels.

Melittin produces a voltage-dependent increase in the conductance of planar lipid bilayers. The conductance increases when the side of the membrane to which melittin has been added (cis-side) is made positive. This paper reports observations on the effect of modifying two positively charged amino acid residues within the NH2-terminal region of the molecule: lysine at position 7 (K7), and the NH2-terminal glycine (G1). We have synthesized melittin analogues in which K7 is replaced by asparagine (K7-N), G1 is blocked by a formyl group (G1-f), and in which both modifications of the parent compound were introduced (G1-f, K7-N). The time required to reach peak conductance during a constant voltage pulse was shorter in membranes exposed to the analogues than in membranes modified by melittin. The apparent number of monomers producing a conducting unit for [K7-N]-melittin and [G1-f]-melittin, eight, was found to be greater than the one for [G1-f], K7-N]-melittin and for melittin itself, four. The apparent gating charge per monomer was less for the analogues, 0.5-0.3 than for melittin, one. Essentially similar results were obtained with melittin analogues in which the charge on K7 or G1 or both was blocked by an uncharged N-linked spin label. These results show that the positive charges in the NH2-terminal region of melittin play a major but not exclusive role in the voltage gating of melittin channels in bilayers.

Voltage-activated cation permeability in high-potassium but not low-potassium red blood cells.

We have recently reported that voltage-activated fluxes of Na, K, and Ca occur in human red blood cells [J.A. Halperin, C. Brugnara, M. Tosteson, T. Van Ha, and D. C. Tosteson. Am. J. Physiol. 257 (Cell Physiol. 26): C986-C996, 1989]. The cation permeability increases progressively as the membrane potential becomes more inside positive above +20 mV. In this paper we show that this effect also occurs in high-potassium (HK), but not in low-potassium (LK), sheep and dog red blood cells. This result suggests that the voltage-activated cation transport pathway is not the result of nonspecific dielectric breakdown of the lipid bilayer but, rather, relates to some membrane component, presumably a protein, that is expressed in HK human and sheep but not in LK sheep and dog red blood cells.

Voltage-activated cation transport in human erythrocytes.

We report here the effects of membrane potential on the permeability of the human erythrocyte to Na, K, and Ca. Membrane potential was changed either by varying the K concentration gradient in the presence of valinomycin or by varying the concentration gradient of the permeant anion nitrate in the presence of 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid. When the membrane potential was changed from inside negative (-10 mV) to inside positive (greater than 40 mV), influx, efflux, and net flux of Na and K increased. Marked net cation loss and cell shrinkage occurred in the absence of a chemical gradient for Na and K. This voltage-dependent increase in Na and K conductance is partially inhibited by 10 microM ruthenium red and persists when the membrane potential is returned to -10 mV after transient exposure to inside-positive potentials. A similar voltage-dependent behavior was found for Ca influx. The voltage-activated Ca influx is almost completely inhibited by 10 microM ruthenium red.

Acid pH induces formation of dense cells in sickle erythrocytes.

When erythrocytes from patients homozygous for hemoglobin S (SS) are swollen or exposed to pH less than 7.40, they lose K, Cl, and water through a volume and pH-dependent KCl cotransport system. We report that carbon monoxide-treated SS cells become progressively denser when incubated for eight to 12 hours in media with pH less than 7.40 (7.3 to 7.0) at constant cell 2,3-diphosphoglycerate (DPG). This phenomenon is maximal in fresh SS cells from the top and middle density fractions, and is absent in cells from the densest fraction. When AA cells are separated according to density, acid pH induces cell shrinkage in the least dense fraction of AA cells, which has considerable KCl cotransport, but produces no change in cell density in the densest fractions of AA erythrocyte, which have no KCl cotransport. These data suggest that dense cells can form in oxygenated SS erythrocytes when the KCl cotransport system is activated by acidification.

Role of chloride in potassium transport through a K-Cl cotransport system in human red blood cells.

In this paper, we report experiments demonstrating the coupling of Cl and K movements in a volume-dependent K-Cl cotransport system in human red blood cells. We show that an outwardly directed Cl gradient can promote net K efflux against an inwardly directed K gradient at constant membrane potential. Red blood cell membrane potential was kept constant by using anions that are not transported through the K-Cl cotransport system but that are more permeable than Cl (NO3 and SCN). Under these conditions, when the activities of band 3 (capnophorin)-mediated anion exchange and of the carbonic anhydrase have been inhibited, it is possible to maintain a Cl gradient at constant membrane potential. Similar data were obtained in human red blood cells (least dense fraction from normal subjects and whole blood from patients with homozygous hemoglobin S disease), in rabbit red blood cells, and in low-K sheep red blood cells. These data confirm that the volume-dependent Cl-dependent K movement in these cells operates through coupled K-Cl cotransport.

Voltage-gated channels formed in lipid bilayers by a positively charged segment of the Na-channel polypeptide.

The Na-channel polypeptide is responsible for the voltage-gated and time-dependent ionic permeability changes that give rise to the action potential in the membranes of nerve cells. We have synthesized a 22-amino acid peptide with a sequence identical to that of the segment named S4, repeat IV of the primary structure of the Na channel. We have found that this peptide induces a voltage- and time-dependent conductance in bilayers formed by a mixture of phosphatidyl-ethanolamine and phosphatidylserine. This conductance is activated when the cis side is made positive, with an apparent gating charge of 3. The results are consistent with the idea that this segment plays a role in determining the voltage sensitivity of the Na channel.

Properties of K+ transport in resealed human erythrocyte ghosts.

We report here our studies on K+ transport in resealed human red cell ghosts (RG) in the presence of 0.1 mM ouabain and 0.01 mM bumetanide, inhibitors of the Na+-K+ pump and Na+-K+-Cl- cotransport, respectively. RG were obtained with the gel-filtration method. K+ efflux from RG was dependent on the pH used in the lysis buffer and increased when the pH used in the lysis buffer and increased when the pH was raised from 5.5 to 8.0. As in intact red cells, RG made from cells of the least dense fraction had a much higher K+ efflux than RG made from cells of the densest fraction. This K+ flux is volume independent and increases when the pH of the flux medium is increased from 6.0 to 8.0. K+ efflux (60-70%) at pH 7.40 from RG made from cells of the least dense fraction is inhibited when Cl- is substituted by nitrate or when the ghosts are resealed in the absence of ATP. This chloride- and ATP-dependent component is markedly reduced in RG made from cells of the densest fraction. An increase in the internal Mg2+ concentration in RG from the least dense fraction induced marked inhibition of K+ efflux. Contrary to intact cells, N-ethylmaleimide (NEM) did not affect K+ efflux from RG. Thus the effects of pH, osmolarity, and NEM on K+ transport in RG are markedly different from those reported in intact erythrocytes.