Evidence that asymmetry of the membrane/cytoskeletal complex in human red blood cell ghosts is responsible for their biconcave shape.

The main conclusion of the results reported in this article is that during centrifugation, sphered red blood cell ghosts become oriented in their attachment to a coverslip such that a dense band within the ghosts lies parallel to the centrifugal field. The result of the orientation of this dense band is that when the attached spherical ghosts are shrunken to become biconcave discs, they do so by directly collapsing on themselves without any lateral motion. This result is interpreted to suggest that a dense band, relative to the dimple, resides in the rim of the ghost and is responsible for its biconcave shape. These results confirm the conclusions reached in a previous publication in which there was the uncertainty that the shape change of the spherical ghosts to discs could not be directly imaged. The present work corrects this limitation by use of a chamber in which the tonicity of the solutions in the ghosts' surround could be altered by perfusion coupled with constant microscopic imaging. The identity of the components that are responsible for the differences in the density (mass) between the rim and the dimple regions of the cytoskeletal/membrane complex in the biconcave disk are unknown. It is also unknown what forces apply or what the explanation is for the unique orientation of the dense band during the ghosts' centrifugation, as described in this article. Nevertheless, the results reported in this article indicate the membrane's underlying cytoskeletal complex is asymmetrically distributed.

Biconcave shape of human red-blood-cell ghosts relies on density differences between the rim and dimple of the ghost's plasma membrane.

The shape of the human red blood cell is known to be a biconcave disk. It is evident from a variety of theoretical work that known physical properties of the membrane, such as its bending energy and elasticity, can explain the red-blood-cell biconcave shape as well as other shapes that red blood cells assume. But these analyses do not provide information on the underlying molecular causes. This paper describes experiments that attempt to identify some of the underlying determinates of cell shape. To this end, red-blood-cell ghosts were made by hypotonic hemolysis and then reconstituted such that they were smooth spheres in hypo-osmotic solutions and smooth biconcave discs in iso-osmotic solutions. The spherical ghosts were centrifuged onto a coated coverslip upon which they adhered. When the attached spheres were changed to biconcave discs by flushing with an iso-osmotic solution, the ghosts were observed to be mainly oriented in a flat alignment on the coverslip. This was interpreted to mean that, during centrifugation, the spherical ghosts were oriented by a dense band in its equatorial plane, parallel to the centrifugal field. This appears to be evidence that the difference in the densities between the rim and the dimple regions of red blood cells and their ghosts may be responsible for their biconcave shape.

Identification of cytoskeletal elements enclosing the ATP pools that fuel human red blood cell membrane cation pumps.

The type of metabolic compartmentalization that occurs in red blood cells differs from the types that exist in most eukaryotic cells, such as intracellular organelles. In red blood cells (ghosts), ATP is sequestered within the cytoskeletal-membrane complex. These pools of ATP are known to directly fuel both the Na(+)/K(+) and Ca(2+) pumps. ATP can be entrapped within these pools either by incubation with bulk ATP or by operation of the phosphoglycerate kinase and pyruvate kinase reactions to enzymatically generate ATP. When the pool is filled with nascent ATP, metabolic labeling of the Na(+)/K(+) or Ca(2+) pump phosphoproteins (E(Na)-P and E(Ca)-P, respectively) from bulk [γ-(32)P]-ATP is prevented until the pool is emptied by various means. Importantly, the pool also can be filled with the fluorescent ATP analog trinitrophenol ATP, as well as with a photoactivatable ATP analog, 8-azido-ATP (N(3)-ATP). Using the fluorescent ATP, we show that ATP accumulates and then disappears from the membrane as the ATP pools are filled and subsequently emptied, respectively. By loading N(3)-ATP into the membrane pool, we demonstrate that membrane proteins that contribute to the pool's architecture can be photolabeled. With the aid of an antibody to N(3)-ATP, we identify these labeled proteins by immunoblotting and characterize their derived peptides by mass spectrometry. These analyses show that the specific peptides that corral the entrapped ATP derive from sequences within ß-spectrin, ankyrin, band 3, and GAPDH.

On the functional use of the membrane compartmentalized pool of ATP by the Na+ and Ca++ pumps in human red blood cell ghosts.

Previous evidence established that a sequestered form of adenosine triphosphate (ATP pools) resides in the membrane/cytoskeletal complex of red cell porous ghosts. Here, we further characterize the roles these ATP pools can perform in the operation of the membrane's Na(+) and Ca(2+) pumps. The formation of the Na(+)- and Ca(2+)-dependent phosphointermediates of both types of pumps (E(Na)-P and E(Ca)-P) that conventionally can be labeled with trace amounts of [gamma-(3)P]ATP cannot occur when the pools contain unlabeled ATP, presumably because of dilution of the [gamma-(3)P]ATP in the pool. Running the pumps forward with either Na(+) or Ca(2+) removes pool ATP and allows the normal formation of labeled E(Na)-P or E(Ca)-P, indicating that both types of pumps can share the same pools of ATP. We also show that the halftime for loading the pools with bulk ATP is 10-15 minutes. We observed that when unlabeled "caged ATP" is entrapped in the membrane pools, it is inactive until nascent ATP is photoreleased, thereby blocking the labeled formation of E(Na)-P. We also demonstrate that ATP generated by the membrane-bound pyruvate kinase fills the membrane pools. Other results show that pool ATP alone, like bulk ATP, can promote the binding of ouabain to the membrane. In addition, we found that pool ATP alone functions together with bulk Na(+) (without Mg(2+)) to release prebound ouabain. Curiously, ouabain was found to block bulk ATP from entering the pools. Finally, we show, with red cell inside-outside vesicles, that pool ATP alone supports the uptake of (45)Ca by the Ca(2+) pump, analogous to the Na(+) pump uptake of (22)Na in this circumstance. Although the membrane locus of the ATP pools within the membrane/cytoskeletal complex is unknown, it appears that pool ATP functions as the proximate energy source for the Na(+) and Ca(2+) pumps.

My passion and passages with red blood cells.

This article mainly presents, in sequential panels of time, an overview of my professional involvements and laboratory experiences. I became smitten with red blood cells early on, and this passion remains with me to this day. I highlight certain studies, together with those who performed the work, recognizing that it was necessary to limit the details and the topics chosen for discussion. I am uncertain of the interest a personal account has for others, but at least it's here for the record.

Directly observed reversible shape changes and hemoglobin stratification during centrifugation of human and Amphiuma red blood cells.

This paper describes changes that occur in human and Amphiuma red blood cells observed during centrifugation with a special microscope. Dilute suspensions of cells were layered, in a centrifuge chamber, above an osmotically matched dense solution, containing Nycodenz, Ficoll, or Percoll (Pharmacia) that formed a density gradient that allowed the cells to slowly settle to an equilibrium position. Biconcave human red blood cells moved downward at low forces with minimum wobble. The cells oriented vertically when the force field was increased and Hb sedimented as the lower part of each cell became bulged and assumed a "bag-like" shape. The upper centripetal portion of the cell became thinner and remained biconcave. These changes occurred rapidly and were completely reversible upon lowering the centrifugal force. Bag-shaped cells, upon touching red cells in rouleau, immediately reverted to biconcave disks as they flipped onto a stack. Amphiuma red cells displayed a different type of reversible stratification and deformation at high force fields. Here the cells became stretched, with the nucleus now moving centrifugally, the Hb moving centripetally, and the bottom of the cells becoming thinner and clear. Nevertheless, the distribution of the marginal bands at the cells' rim was unchanged. We conclude that centrifugation, per se, while changing a red cell's shape and the distribution of its intracellular constituents, does so in a completely reversible manner. Centrifugation of red cells harboring altered or missing structural elements could provide information on shape determinants that are still unexplained.

Fluorescent imaging of Cl- in Amphiuma red blood cells: how the nuclear exclusion of Cl- affects the plasma membrane potential.

In this work, we test the idea that most, if not all, cellular Cl- of Amphiuma red blood cells is contained in the cytoplasm. If true, this could resolve the difference between the measured plasma membrane potential (Em) and that expected from the Donnan equilibrium distribution of Cl-. We studied the changes in the fluorescence intensity of the Cl- -sensitive dye, MQAE, entrapped in red cells that occurred when intracellular Cl- was exchanged with NO3-. We could thus monitor the distribution of Cl- between the nuclear and cytoplasmic compartments. We found that essentially all of the cell's Cl- resides in the cytoplasm. Knowing the volume of the cell occupied by the nucleus, we could accordingly correct the measured values of cell Cl-. This resulted in establishing a concordance between the measured values of Em and those calculated from the corrected values of the Cl- ratio, thus explaining the discrepancy. The exclusion of Cl- from the nucleus may result from its unusually high content of "excess" DNA that imposes an imbalance of net negative charge.

Tetrodotoxin-sensitive Na+ channels and muscarinic and purinergic receptors identified in human erythroid progenitor cells and red blood cell ghosts.

This article concerns the identification of different types of voltage-gated Na(+) channels and of muscarinic and purinergic receptors that are expressed in human erythroid precursor cells and red cell ghosts. We analyzed, by RT-PCR, RNA that was extracted from purified and synchronously growing human erythroid progenitor cells, differentiating from erythroblasts to reticulocytes in 7 to 14 days. These extracts were free of white cell and platelet contamination. Two types of voltage-gated, tetrodotoxin-sensitive Na(+) channels were found. These were Na(v)1.4 and Na(v)1.7, the former known to be present in skeletal muscle and the latter in peripheral nerve. By using a pan Na(+) channel antibody and Western blotting, an immunoreactive channel was detected in ghosts of human red blood cells, consistent with the expression of these two channels. The transcripts for four of the five known subtypes of muscarinic receptors were also identified, including subtypes M2, M3, M4, and M5, whereas subtype M1 was not found. Expression was also detected for the purinergic type receptors P2X(1), P2X(4), P2X(7), and P2Y(1) whereas types P2Y(2), P2Y(4), and P2Y(6) were not found. We also searched for but did not find transcripts for hBNP-1, a type 1b human brain sodium phosphate cotransporter, and cystic fibrosis transmembrane conductance regulator (CFTR). Implications regarding the presence of these different types of channels and receptors in human red blood cells and their functional significance are discussed.

The hSK4 (KCNN4) isoform is the Ca2+-activated K+ channel (Gardos channel) in human red blood cells.

The question is, does the isoform hSK4, also designated KCNN4, represent the small conductance, Ca2+-activated K+ channel (Gardos channel) in human red blood cells? We have analyzed human reticulocyte RNA by RT-PCR, and, of the four isoforms of SK channels known, only SK4 was found. Northern blot analysis of purified and synchronously growing human erythroid progenitor cells, differentiating from erythroblasts to reticulocytes, again showed only the presence of SK4. Western blot analysis, with an anti-SK4 antibody, showed that human erythroid progenitor cells and, importantly, mature human red blood cell ghost membranes, both expressed the SK4 protein. The Gardos channel is known to turn on, given inside Ca2+, in the presence but not the absence of external Ko+ and remains refractory to Ko+ added after exposure to inside Ca2+. Heterologously expressed SK4, but not SK3, also shows this behavior. In inside-out patches of red cell membranes, the open probability (Po) of the Gardos channel is markedly reduced when the temperature is raised from 27 to 37 degrees C. Net K+ efflux of intact red cells is also reduced by increasing temperature, as are the Po values of inside-out patches of Chinese hamster ovary cells expressing SK4 (but not SK3). Thus the envelope of evidence indicates that SK4 is the gene that codes for the Gardos channel in human red blood cells. This channel is important pathophysiologically, because it represents the major pathway for cell shrinkage via KCl and water loss that occurs in sickle cell disease.

Na pump isoforms in human erythroid progenitor cells and mature erythrocytes.

This study is aimed at identifying the Na pump isoform composition of human erythroid precursor cells and mature human erythrocytes. We used purified and synchronously growing human erythroid progenitor cells cultured for 7-14 days. RNA was extracted from the progenitor cells on different days and analyzed by RT-PCR. The results showed that only the alpha1, alpha3, beta2, and beta3 subunit isoforms and the gamma modulator were present. Northern analysis of the erythroid progenitor cells again showed that beta2 but not beta1 or alpha2 isoforms were present. The erythroid cells display a unique beta subunit expression profile (called beta-profiling) in that they contain the message for the beta2 isoform but not beta1, whereas leukocytes and platelets are known to have the message for the beta1 but not for the beta2 isoform. This finding is taken to indicate that our preparations are essentially purely erythroid and free from white cell contamination. Western analysis of these cultured progenitor cells confirmed the presence of alpha1, alpha3, (no alpha2), beta2, beta3, and gamma together now with clear evidence that beta1 protein was also present at all stages. Western analysis of the Na pump from mature human erythrocyte ghosts, purified by ouabain column chromatography, has also shown that alpha1, alpha3, beta1, beta2, beta3, and gamma are present. Thus, the Na pump isoform composition of human erythroid precursor cells and mature erythrocytes contains the alpha1 and alpha3 isoforms of the alpha subunit, the beta1, beta2, and beta3 isoforms of the beta subunit, and the gamma modulator.