Joseph F. Hoffman

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 \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}{\mathrm{K}}_{{\mathrm{o}}}^{+}\end{equation*}\end{document} and remains refractory to \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}{\mathrm{K}}_{{\mathrm{o}}}^{+}\end{equation*}\end{document} 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°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.

The membrane locus of Ca-stimulated K transport in energy depleted human red blood cells.

SummaryEnergy depleted human red cells subsequently exposed to iodoacetate (IAA) develop, upon the addition of Ca, a marked increase in K permeability, while Na permeability is unaltered. The kinetic characteristics of this augmented K permeability indicate that the transport process is membrane mediated. Thus, the inward and outward rate constants for K increase as the concentration of external K is increased reaching maximum values between 2 to 5mm; further increases in external K results in a partial reduction of the rate constants. In addition, the Ca-stimulated K transport system displays counterflow of42K during its influx when a large gradient of39K exists (inside high, outside low). Furthermore, the Ca-induced K transport is inhibited by ouabain. The sensitivity of the Ca-induced system to ouabain parallels the action of ouabain on the Na−K pump. At least part of the increased K transport occurs through a preexisting pathway since ouabain bound to cells before exposure to Ca and IAA results in an inhibition of K outflux. Since ouabain does not alter the affinity of the cells for Ca, it is concluded that at least a portion of the increased K transport results from Ca acting to increase the turnover rate of the same system which serves as the Na−K pump in normal red cells.

Ca-induced K transport in human red cells: Localization of the Ca-sensitive site to the inside of the membrane

Summary The differential effects of internal and external Ca on increasing the membrane permeability to K (but not to Na) have been studied in ghosts made from energy depleted red cells. All changes in K transport induced by Ca were found to be oligomycin-sensitive. Internal Ca was found to be much more effective than external Ca in stimulating K transport. External EGTA was more effective in reversing the action of external Ca than in reversing the action of internal Ca. In contrast, internal EGTA made the ghost completely refractory to external Ca. Thus it appears that internal Ca is necessary for activation of K transport in energy depleted cells.

Interactions of the fluorescent anion 1-anilino-8-naphthalene sulfonate with membrane charges in human red cell ghosts

SummaryThe interaction of the fluorescent anion 1-anilino-8-naphthalene sulfonate (ANS) with erythrocyte membranes was studied as a function of the charges in the membrane. The membrane charges were altered by including in the incubation medium the organic ions tetraphenylboron (TPB−), tetraphenylarsonium (TPA+) and tetraphenylphosphonium (TPP+) which differ in charge but have similar structure and high affinity for the membrane. TPB− decreases whereas TPA+ and TPP+ increase ANS fluorescence arising from the membrane. The fluorescence changes are accompanied by variations in the affinity and capacity of the membrane for ANS. The fluorescence arising under conditions where all the ANS present is within the membrane (by extrapolation to infinite membrane concentration) does not vary with these ions. Salicylate and SCN− which inhbit anion permeability also decrease ANS fluorescence with no apparent change in quantum yield. These results indicate that ANS exists as an anion in the membrane, associated with the regions involved in ion permeability, and that the concentration of ANS in the membrane is a function of the concentration of all the ions present in the medium and their relative affinities for the membrane. Thus, variations in ANS fluorescence reflect redistributions of the ANS ions following changes in electrostatic interactions with the membrane fixed charges.

On red blood cells, hemolysis and resealed ghosts.

This paper aims to consider, with a historical perspective, the characteristics of red blood cells and their ghosts that are associated with osmotic hemolysis and its reversal. Since hemolysis refers to the process by which a cell becomes permeable to hemoglobin (Hb) the term “ghost” is used to describe the resultant envelope or cell-like structure (also referred to as post-hemolytic residue or stroma) that survives the transition. This definition of the term, ghost, emphasizes the functional involvement of the plasma membrane and is independent of, or at least not biased by, the circumstances leading to its production. This definition, based on a loss of Hb by a change in the membrane’s permeability, excludes the types of changes in a cell’s Hb content that occurs, for instance, during erythroid maturation or by age related changes in cell density. While this definition of a ghost is general and independent of Hb content (ghosts can range from being nominally Hb-free to containing almost the cell’s original amount), it should also be understood that the resultant types/properties of ghosts reflect the conditions that attended the hemolytic step(s) and any subsequent treatment(s). This means that ghost characteristics are method-dependent and should be specified in every case.

The interaction of fluorescent probes with anion permeability pathways of human red cells

SummaryThe fluorescent probe ANS is a permeant anion in human red cells. The rate of ANS permeation is decreased by lyotropic anions and increased by low ionic strength, resembling the response of Cl and SO4 transport to changes in the composition of the medium. ANS inhibits Cl and SO4 exchange measured at 0 and 37°C, respectively. The inhibitory potency of ANS isomers increases in the sequence 1,8 ANS<2,8 ANS<1,4 ANS<2,6 TNS<5,2 ANS. The disulfonic stilbene derivative SITS inhibits Cl exchange 50%. Combinations of ANS and SITS result in additive inhibitory effects regardless of the ANS concentration. Combinations of dipyridamole and ANS show additive inhibitory effects only at low concentrations of the latter. The mechanisms of inhibition by ANS are discussed in terms of (1) interactions between the probe and an anion carrier and (2) modifications of the membrane surface charge by ANS. Assuming that ANS bound to the membrane surface produces a negative surface charge, ANS-dependent surface potentials of magnitude sufficient to account for the observed inhibition can be calculated using double layer theory. It is suggested that anionic amphiphiles inhibit anion and increase cation permeability through modifications of surface charge that alter the ion concentrations at the permeability barriers and a second step, affected by SITS, is involved in anion permeation.

Interrelations among Na and K content, cell volume, and buoyant density in human red blood cell populations

SummaryThis study establishes a method for determining the concentration of Na and K in single red blood cells from electron probe microanalysis of a cells Na and K content. To this end, red blood cells were separated into subpopulations according to their buoyant density by means of bovine serum density gradient centrifugation. Cell water and Na+K contents were then determined in each fraction by conventional analytic methods with cell volume estimated from measurements of hematocrits and cell number. It was found that an inverse relationship obtains between the mean cell volume and buoyant cell density since cells increased in size as density decreased. Although the amount of hemoglobin per cell was found to slightly increase as cell density decreased, hemoglobin concentration showed the inverse relationship, indicating that buoyant cell density differences are primarily the result of differences in hemoglobin concentration. In confirmation of Funder and Wieth (Funder, J., Wieth, J.O. 1966.Scand. J. Lab. Invest.18:167–180) cell water and cell volume was found to vary directly with the summed content of Na+K. Finally, by means of electron probe microanalysis of single cells, the cellular concentration of hemoglobin was found to vary inversely with the Na+K content, providing a quantitative basis for directly estimating cell volume, and thus ionic concentration, with this technique.

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 α1, α3, β2, and β3 subunit isoforms and the γ modulator were present. Northern analysis of the erythroid progenitor cells again showed that β2 but not β1 or α2 isoforms were present. The erythroid cells display a unique β subunit expression profile (called β-profiling) in that they contain the message for the β2 isoform but not β1, whereas leukocytes and platelets are known to have the message for the β1 but not for the β2 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 α1, α3, (no α2), β2, β3, and γ together now with clear evidence that β1 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 α1, α3, β1, β2, β3, and γ are present. Thus, the Na pump isoform composition of human erythroid precursor cells and mature erythrocytes contains the α1 and α3 isoforms of the α subunit, the β1, β2, and β3 isoforms of the β subunit, and the γ modulator.

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 Ca2+ 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 Ca2+ pump phosphoproteins (ENa-P and ECa-P, respectively) from bulk [γ-32P]-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 (N3-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 N3-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 N3-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 β-spectrin, ankyrin, band 3, and GAPDH.

Candida epiglottitis in an adult with acute nonlymphocytic leukemia

A 40-year-old white woman presented with fever, otalgia, and odynophagia and was found to have a peripheral white blood cell count of 90,000/mm3. A diagnosis of acute myelogenous leukemia was made. Further evaluation of symptoms and source for fever led to the diagnosis of Candida albicans epiglottitis. This is the first reported case of fungal epiglottitis in an immunocompromised adult.