Referência : http://www.time.com/time/time100/scientist/profile/fleming.html Alexander Fleming A spore that drifted into his lab and took root on a culture dish started a chain of events that altered forever theThe improbable chain of events that led Alexanderbacteriologist." Although he went on to performFleming to discover penicillin in 1928 is the stuff ofadditional experiments, h
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Nehrkelab.comThe hSK4 (KCNN4) isoform is the Ca2؉-activated K؉
channel (Gardos channel) in human red blood cells
Joseph F. Hoffman*†, William Joiner*‡, Keith Nehrke§, Olga Potapova*¶, Kristen Foye*ʈ, and Amittha Wickrema**
*Departments of Cellular and Molecular Physiology and Pharmacology, Yale University, New Haven, CT 06520; §Department of Medicine, University ofRochester Medical Center, Rochester, NY 14642; and **Section of Hematology͞Oncology, University of Chicago, Chicago, IL 60637 Contributed by Joseph F. Hoffman, April 21, 2003 The question is, does the isoform hSK4, also designated KCNN4,
cells allow us to study purely erythroid developmental stages free represent the small conductance, Ca2؉-activated K؉ channel (Gar-
of contamination from white cells or platelets. This conclusion dos channel) in human red blood cells? We have analyzed human
is based on ‘‘␤ profiling,’’ by which we mean that the progenitor reticulocyte RNA by RT-PCR, and, of the four isoforms of SK
cells were found to contain the message for the ␤2 and not the channels known, only SK4 was found. Northern blot analysis of
␤1 isoforms of the ␤ subunit of the Na pump in contrast to a purified and synchronously growing human erythroid progenitor
white cell and platelet library that contained the message for the cells, differentiating from erythroblasts to reticulocytes, again
␤1 but not the ␤2 isoforms (20). Our studies using progenitor showed only the presence of SK4. Western blot analysis, with an
cells indicate that SK4 is the isoform that subserves the functions anti-SK4 antibody, showed that human erythroid progenitor cells
of the Gardos channel in human red blood cells. This identifi- and, importantly, mature human red blood cell ghost membranes,
cation is based on RT-PCR, Northern and Western blotting of both expressed the SK4 protein. The Gardos channel is known to
progenitor cells, and Western analysis of human red blood cell turn on, given inside Ca2؉, in the presence but not the absence of
ghosts. We also found that SK4, when heterologously expressed external K؉
in Chinese hamster ovary (CHO) cells, displays functional o and remains refractory to Ko added after exposure to
inside Ca2؉. Heterologously expressed SK4, but not SK3, also
characteristics of the Gardos channel similar to those seen in 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,
Reticulocyte and Erythroid Progenitor Cell RNA. This study used
samples of RNA taken from the same preparations of reticulo- as are the Po values of inside–out patches of Chinese hamster ovary
cytes and human erythroid progenitor cells purified and cultured cells expressing SK4 (but not SK3). Thus the envelope of evidence
as described previously (20). All of these preparations were indicates that SK4 is the gene that codes for the Gardos channel in
found to be free of leukocyte and platelet contamination based human red blood cells. This channel is important pathophysiologi-
on the criterion of ‘‘␤ profiling,’’ as previously established (20).
cally, because it represents the major pathway for cell shrinkage
RT-PCR was carried out by using the SK isoform specific via KCl and water loss that occurs in sickle cell disease.
primers given in Table 1. Note that one primer pair, labeled SK1–3, was designed to detect these three channel isoforms but Four isoforms (SK1–4) of the small conductance Ca2ϩ- not SK4. To confirm that primers were capable of amplifying a
activated Kϩ channel have been identified (1–5). These specific product, RT-PCR was performed with each set of channels, with acronyms small conductance (SK), intermediate primers and plasmids individually encoding each of the channel conductance (IK), and potassium channel calcium-activated isoforms. In each case, a product of the expected size was intermediate͞small conductance subfamily N (KCNN), are amplified with Ͼ96% sequence identity.
highly conserved Ca2ϩ-activated inward rectifiers (see refs. 1–5).
Although KCNN is the notation assigned by GenBank (6), we Northern Blotting. Northern blotting was carried out as described
use herein the SK notation and report studies that deal mainly before (20). DNA probes were derived from the following with the human isoform, i.e., hSK4, recognizing that there is a gel-purified DNA templates: a 436-bp BamHI–HindIII fragment parallel with the rat, rSK4, and mouse, mSK4, isoforms (7). We of rat SK1 (rSK1); a 582-bp EcoRV–NarI fragment of rat SK2 are principally concerned with SK4 because of its putative (rSK2); a 710-bp SmaI–SmaI fragment of rat SK3 (rSK3); and identification as the Ca2ϩ-activated Kϩ channel, referred to as a 723-bp SphI–SfiI fragment of human SK4 (hSK4). These DNA the Gardos channel (8), in human red blood cells. Activation of fragments were radiolabeled by using a Prime-It II kit (Strat- the channel results in a marked hyperpolarization of the mem- agene) and 32P-dCTP and purified on G-50 Spin Columns brane accompanied by shrinkage of the cell due to the loss of KCl (Roche Applied Science, Indianapolis) to achieve a specific and water. The main reason the Gardos channel has been activity of Ϸ109 dpm͞g. As a positive control, probes were assigned to the SK4 gene in human red blood cells is because of also hybridized with a human multiple tissue Northern blot parallels in its electrophysiological characteristics (7, 9, 10) between the intact cell and the expressed channel as well as its pharmacological sensitivities; the channel is inhibited by charyb- Western Blotting. The anti-mouse SK4 (mSK4͞IKCa) antibody (21)
dotoxin (11) and clotrimazole (CLT) (12) but not by apamin was custom produced by Research Genetics, a division of Invitro- (13). The SK4 expressed in different cell types shares this inhibitory profile (1, 7). In sharp contrast, expressed SK1, 2, or 3 channels are inhibited by apamin (14–17) but not by CLT (18).
Abbreviations: CLT, clotrimazole; CHO, Chinese hamster ovary.
Obviously, it is necessary to go beyond the foregoing correlations †To whom correspondence should be addressed at: Department of Cellular and Molecular of channel characteristics to establish which isoform(s) of the SK Physiology, Yale University School of Medicine, 333 Cedar Street, New Haven, CT 06520-8026. E-mail: email@example.com.
family are actually found in human red blood cells.
‡Present address: Department of Neuroscience, Howard Hughes Medical Institute, 232 Our approach exploits the use of human erythroid progenitor Stemmler Hall, University of Pennsylvania, Philadelphia, PA 19104.
cells in which we have previously determined the subunit types ¶Present address: Department of Molecular Biophysics and Biochemistry, Yale University, and isoform composition of the endogenously expressed Naϩ pumps (19, 20). Our particular preparations of the progenitor ʈPresent address: Genaissance Pharmaceuticals, New Haven, CT 06511.
7366 –7371 ͉ PNAS ͉ June 10, 2003 ͉ vol. 100 ͉ no. 12
www.pnas.org͞cgi͞doi͞10.1073͞pnas.1232342100 Table 1. PCR primer pair sequences (all designed against human
where they were Ca2ϩ-dependent and displayed the expected sequences) that were selected to have distinct and high
unitary conductance characteristics (1).
specificity for each of the indicated SK isoforms of the
The pipette solution for whole-cell experiments consisted of Ca؉؉-activated K؉ channel
(in mM): 30 KCl, 100 K-gluconate, 5 EGTA, and 10 Hepes (pH 7.2). This solution was supplemented with 4.27 or 4.74 mM CaCl2 to achieve free Ca2ϩ concentrations of 1.0 or 3.0 M, respec- tively. All whole-cell recordings were performed by using 200-ms ramps from Ϫ120 to ϩ80 mV from a holding potential of Ϫ70 mV. Current densities were measured for each cell by dividing the current amplitude at 60 mV by the capacitance.
For inside-out patch recordings of stably transfected cells, pipettes were filled with (in mM): 30 KCl, 100 K-gluconate, 1 MgCl2, and 10 Hepes (pH 7.2), and the cytoplasmic side of the membrane was perfused with (in mM): 30 KCl, 100 K-gluconate, 5 EGTA, 10 Hepes (pH 7.2), and 4.27 CaCl2 (1 M free Ca2ϩ).
In some experiments, lyophilized thioredoxin peroxidase (23), i.e., calpromotin (24, 25), was dissolved in the latter solution at a concentration of 10 M and used to perfuse excised inside-out patches. All patch recordings were made over a period of 30–60 s at a holding potential of Ϫ80 mV, first at 25°C and subse- quently, after rapidly switching (Ͻ10 s), at a controlled bath temperature of 35°C in a temperature-controlled 35-mm tissue culture cup. Electrical heating was mediated via a Pt͞Ir oxide film on the outside of the cup (Bioptechs, Butler, PA).
and 86Rb؉ Fluxes. Blood was drawn into heparin from
In the case of the SK1–3 (i.e., SK1, SK2, and SK3) primer set, M ϭ A or C; R ϭ normal volunteers and used without delay. Net Kϩ effluxes were measured by incubating red cells in a low Kϩo medium for various time periods. The Gardos channel (i.e., Ca2ϩ-activated Kϩ gen. The antibody was directed toward an mSK4-specific peptide channel) was activated, in the presence of Ca2ϩ with the amino acid sequence RQVRLKHRKLTEQVNSMVD.
the divalent ionophore, A23187, or energy depletion or both, as Pellets of 7- and 13-day-old cultures of human erythroid progenitor indicated below. The experimental protocols used were varia- cells (see ref. 20) as well as human brain, kidney, and parotid tissues tions on those described by others (26, 27). Because details of the were prepared by previously described methods (see ref. 21).
protocols varied, we have adopted abbreviations for some con- Human red blood cell ghosts were prepared from heparinized stituents that were common to many of the solutions used. Thus, peripheral blood by hypotonic lysis, as described (22), and were H is Hepes buffer, C is CaCl2, M is 0.2 mM MgCl2, A is 10 M frozen, thawed, and washed before use.
A23187, V is 1.0 mM orthovanadate, I is 10 mM inosine, IA is Approximately10 g of crude protein was separated by two- 6.0 mM iodoacetamide, and CLT is 10 M chlotrimazole. It phase Tricine polyacrylamide gel electrophoresis (10% T͞6% C should be understood that V is used to inhibit the Ca2ϩ pump, resolving layer, 4% T͞3% C stacking layer) and transferred onto thereby allowing for the accumulation of (Cai ) necessary to poly(vinylidene difluoride) membrane (BioDyne PVDF, Pall activate the Gardos channel. The combination of I plus IA is Filtration, General Electric) in buffer containing 10 mM 3- used to deplete the cells of energy (ATP) that could interfere [cyclohexylamino]-1-propanesulfonic acid (CAPS) adjusted to with the activation of the Gardos channel. In all experiments, pH 11 and 10% methanol. Blotting was then carried out as final incubations were carried out in the absence and presence described (21). To assess specificity, the mSK4 antibody was of CLT, and all samples taken from suspensions where CLT was preincubated for 1 h with a 50-fold molar excess of competitor absent were mixed before centrifuging with a stop solution containing CLT. This procedure provided the time resolution for peptide corresponding to the epitope recognition sequence rapid fluxes. Hematocrits for net Kϩ measurements varied from 11% to 20%. The pH of all solutions was Ϸ7.4 at 37°C. Changing the temperature from 37 to 27°C lowers the pH by Ϸ0.1 pH unit, Stable SK-Expressing Cell Lines. CHO cells stably expressing hSK4
without appreciable effect on the Kϩ flux (28). For Table 2, the channels as described (1) were used in this study. Another CHO following experimental conditions were used. Experiment A: cell line stably expressing rSK3 was established by similar means, cells were washed with a solution containing (in mM) 40 NaCl, except that the cDNA was carried in the plasmid pcDNA 3.1 Zeo 90 NaSCN, 2 KCl, M, and 20 H. The cells were then energy (from Guy Moss, University College, London), and Zeocin depleted by incubation in this solution together with 1.3 mM C, (Invitrogen) was used to select stable transfectants. Stably I, IA, and V for 25 min at 37°C. The suspension was then split, transfected survival colonies were sorted by FACS as described with half incubated at 27°C and the other half at 37°C. After 3 (1). A single clonal population of cells was used for each cell line.
min, samples were taken at 0, 15, 30, 45, and 60 min. Experiment B: cells were washed in the presence or absence of SCNϪ in Electrophysiology of SK Channels. Single-channel recordings were
solutions containing (in mM): either 40 NaCl ϩ 90 NaSCN or performed by using the methods described (1). For whole-cell 130 NaCl together with 0.5 KCl, 30 H, and M. After washing, the recordings, the standard bath solution (5 mM Ko) consisted of cells were suspended, respectively, in each of these solutions (in mM): 140 NaCl, 1.0 CaCl2, 5 KCl, 29 glucose, 25 Hepes (pH together with the additions of 1.3 mM C, I, IA, and V. The cells 7.4). Bath composition for ion substitution experiments was were then energy depleted by incubation in these solutions for 25 min at 37°C. Then each solution was split, with half incubated at 0 mM Kϩo. It should be understood that the Kϩ channels studied 27°C and the other half at 37°C. After 3 min, aliquots were taken in CHO cells were seen not in control cells but only in cells that over a 30-min period for determination of Kϩ PHYSIOLOGY
had been transfected with either the rSK3 or hSK4 isoforms, cells were energy depleted by incubation for 3 h in a solution PNAS ͉ June 10, 2003 ͉ vol. 100 ͉ no. 12 ͉ 7367
Table 2. The effect of temperature on the net efflux of K؉ from
human red blood cells after activation of the Gardos channel by
The SK4 isoform of the Ca2ϩ-activated Kϩ channel is present in human The effluxes of Kϩ were measured at either 27 or 37°C (see Methods for reticulocytes. PCR products were obtained with the isoform-specific primers details) and estimated from the rate of increase in Kϩ defined in Table 1. Single-stranded cDNA derived from reticulocytes (see o medium. The cells were treated with V (except Experiment C) to Methods) was used as template. Lanes 1 and 2 had, respectively, 25- and 4-l inhibit Caϩϩ efflux via the Caϩϩ pump. In addition, the cells were energy samples applied to the gel. Lane 3 is a water control. The mass ladder (in base depleted in Experiments B and C but not in A, in which they were normal. Kϩ pairs) is shown at left (Life Technologies, Grand Island, NY). The expected efflux from cells were begun in Experiments A and B after a 3- to 5-min product size was 1,767, and a product of approximately this size was found in equilibration at their respective temperatures, and in Experiment C, after lanes 1 and 2. The product was sequenced in this and other analyses and was addition of the ionophore A23187 (A23). The Kϩ shown to have Ͼ98% identify with the expected sequence for SK4.
o (mM) values refer to time zero. The effluxes of Ki were also estimated from the rate of appearance of medium 86Rb from cells preloaded with 86Rb(Experiment D). The cells were treated with V to inhibit Caϩϩ efflux via the analogous results with the second set of SK4 primers given in Caϩϩ pump. In addition, the cells were energy depleted. 86Rb effluxes, from Table 1 (data not shown). Use of any of the other isoform cells having been washed in the cold, were begun after a 3- to 5-min equili-bration of the cell suspensions at their respective temperatures. All effluxes primers listed in Table 1 for SK1, 2, or 3 was negative with regard were carried out in the absence and presence of CLT and presented as the to their specific presence in these RNA preparations. When CLT-sensitive flux (⌬CLT). The CLT-insensitive efflux was in all cases Ͻ5–10% of products did appear, they were neither of the expected size nor efflux (Experiments A–C) appeared to be did they display any sequence homology with the isoform being exponential in all cases, the outward rate constant, °k⌬CLT tested. Because we used preparations of reticulocytes that were mated from the initial rate, as explained in Methods. The values of °k⌬CLT free from white cell and platelet contamination by the criterion Experiment D represent the means Ϯ SEM, where n ϭ 4.
of ‘‘␤ profiling’’ (20), the results strongly indicated that SK4 was the isoform responsible for the Gardos channel in human containing (in mM): 10 NaCl, 50 KCl, 60 KSCN, 1.0 Na erythrocytes. However, there is a caveat to this interpretation, 30 H, I, IA, and V. The cells were washed and resuspended in because SK4 has also been identified as a constituent of human the following solution (in mM): 60 NaCl, 60 NaSCN, 0.4 KCl, 30 lymphocytes (3). Thus, although contamination of our prepara- H, M, I, IA, and V together with 50 M C. After 3 min at either tions is improbable, it must remain a possibility.
27 or 37°C, the flux was initiated by addition of A with samples We next turned to our preparations of human erythroid taken at 0, 45, 90, 135, and 180 s. Experiment D: Cells loaded progenitor cells, because, as shown before (20), an erythroid ‘‘␤ with 86Rb by incubation for 3 hr in (in mM): 30 KCl, 90 KSCN, profiling’’ was also a characteristic of these differentiating cells.
20 H and M. Cells were then resuspended in this solution Fig. 2 shows the results of Northern analysis of these progenitor together with 1.3 mM C, I, IA, and V and incubated at 37°C for cells from days 7–12 of culture. The evidence presented in Fig.
15 min before washing in the cold. Cells were resuspended at 2 shows that SK4 is present, and there is a clear tendency for its 27°C and 37°C and after 3 min samples were taken at 0, 5, 10, 15, expression level to increase with increasing maturation. The positive control blots are given on the right side of Fig. 2 and Calculation of 86Rb rate constants were the same as previously made use of mRNA from human placental tissue, which is known described (29). Hematocrits (Hcts) were calculated by using the (17) to contain the SK4 isoform (1, 17). Although we saw no equation, Hct ϭ (SWB Ϫ So)͞(Si Ϫ So), where SWB is the whole evidence by Northern analysis for SK1 or SK3, some blots blood, cellular (Si), or supernatant (So) concentration of Kϩ. The indicated that hSK2 might be present. This was not pursued outward rate constants (hrϪ1) calculated for the results in Table because of failure to find the SK2 isoform by RT-PCR and because the Gardos flux in human erythrocytes is not inhibited Ro͞[Kϩ]i, where Ro (in mM͞unit time) is the initial rate assuming by apamin (13). Thus we conclude from Northern analysis that the curves are single exponentials, and [Kϩ]i is in mmol͞l cell water. The percent water is taken from the difference in wet and dried weights of packed cells. CLT, inosine, and iodoacetamide were obtained from Sigma; A23187 from Calbiochem; and 1,2-bis(2-aminophenoxy)ethane-N,N,NЈ,NЈ-tetraacetate or -tetraacetic acid–acetoxymethyl ester (BAPTA-AM) from Mo- lecular Probes. All other chemicals, wherever possible, were of Results
Our prime aim in undertaking the studies reported here was to
Northern blots probed for the mRNA encoding the Ca2ϩ-activated Kϩ identify the gene that codes for the Gardos channel (or Ca2ϩ- channel isoform, SK4, using RNA prepared from cultured human erythroid dependent K permeability) of human red blood cells. We first progenitor cells at different stages of maturation (see Methods for details).
screened, by RT-PCR, our previous preparations of RNA ex- Also shown is the positive control blot for SK4 (Right), where H is heart; B, tracted from human reticulocytes (19) by use of the primer sets brain; P, placenta; L, lung; Li, liver; S, skeletal muscle; K, kidney; and Pa,pancreas, all from human mRNA. These results parallel our previous finding listed in Table 1, to establish which isoforms of the SK (or that SK4 is present in reticulocytes. Note that SK4 is present in the progenitor KCNN) channel family were present. As is evident in Fig. 1, the cells at day 7 increasing with differentiation through day 12. The transcript isoform SK4 is present in these preparations. We also obtained 7368 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.1232342100
Western blots showing that the protein for the Gardos channel isoform, SK4, is present in cultured human erythroid progenitor cells and inghost membranes made from mature human red blood cells. The antibodywas prepared against an SK4-specific peptide and used as described in Meth-ods. The two positive controls are human parotid gland (P) and kidney (K) withthe negative control being brain (B). It is clear that a band of the appropriatemolecular weight is present in the human erythroid progenitor cells as theymature from days 7 to 13. It is also evident that the SK4 band is present inhuman red cell ghost membranes (RBC). The decrease in the blot intensity ofthe D13 band compared with D7 is primarily due to the decreased proteincontent (cell number) of cells loaded onto the gel. The slight variation in themolecular weights of SK4 bands seen in the progenitor cells, relative to theother bands, may be due to posttranslational modification or higher saltconcentration in the loading mixture. It should also be mentioned that, exceptin the parotid lane, there are higher molecular weight bands (not shown) thatin each case react with the antibody. Importantly, preincubation of theantibody with purified peptide that contains the antigenic epitope producesa complete loss of reactivity in all lanes except in brain, where it is muchreduced, and in ghosts, where it is only faintly present in the highest molecularweight bands (data not shown).
differentiating human erythroid progenitor cells expresses only We also performed a Western blot analysis, the results of which are shown in Fig. 3. By using an antibody directed against a peptide whose sequence is derived from mSK4, we find that a o on the activity of heterologously expressed protein of the appropriate molecular weight is detected not only SK3 and SK4 channels in CHO cells. Cells were washed twice in 0 mM Ko and in the human erythroid progenitor cells but also in mature then incubated with 100 M 1,2-bis(2-aminophenoxy)ethane-N,N,NЈ,NЈ-tetraacetate or -tetraacetic acid–acetoxymethyl ester (BAPTA-AM) in either 5 human erythrocyte membranes (21, 30). This signal was com- mM Kϩ (open bars) or 0 mM Kϩ (dark bars) containing media for 3–5 h at room pletely competed through preincubation of the antibody with the temperature (see Methods). At the end of this period, cells were perfused with antigenic peptide, demonstrating specificity of the interaction.
incubation solution before and during seal formation, breakthrough, and Another indication of the specificity of the SK4 antibody is that achievement of whole-cell recording mode. Current amplitude at 60 mV was it showed no crossreactivity with brain, a tissue known to express then measured for each cell first during continued perfusion with incubation SK1, 2, and 3 but not the SK4 isoform.
solution and then after switching from 0 to 5 Ko or vice versa. The whole-cell We next compared several functional characteristics of the currents in expressed SK3 channels (A) were insensitive to the presence or Gardos channel in human erythrocytes with the behavior of SK4 absence of Ko , whereas the currents in expressed SK4 channels (B) weremarkedly diminished by preincubation in 0 mM Kϩ. (C) The decrease in expressed in transfected cells. The first modality to be examined whole-cell currents that occurred during the first 2 h of incubation of SK4 expressing cells in 0 mM Ko . (D) SK4 cells can recover from incubation in 0 mM It is known from previous work (9, 31) that for the Gardos o by reexposure to 5 mM Ko over this time period. SK4 cells exposed for longer periods of time (B, dark bars) to 0 mM Kϩ remain refractory to 5 mM Kϩ.
channel at the time that the cytoplasmic surface of the channel͞ Error bars represent Ϯ SEM, where n ϭ 9 –15 separate observations with SK3 (A) and 11–15 for SK4 (B); in C and D, n ϭ 3–12.
is introduced inside and, at least in human red cell ghosts (9, 31), will not open even when Kϩo is subsequently over this same time period. Thus, SK4 channels expressed in added. We wished to test whether SK3 or SK4 channels heter- CHO cells behave like the Gardos channel in intact human ologously expressed in CHO cells displayed a similar depen- erythrocytes and ghosts because they stay closed in the absence dence on Kϩo. To this end, stably transfected CHO cells were of Kϩ but differ from them in that the channels are able to open preincubated for varying periods of time in the presence or after the addition of Kϩ. The basis for this difference between o . Whole-cell recordings were then made, with Cai expressed SK4 channels and Gardos channels is not known, but being introduced with a patch pipette, as described in Methods it should be remembered that, because these patched CHO cells and the Fig. 4 legend, with subsequent determination of the are attached to the substrate, Kϩ trapped in the cell͞substrate current density. As shown in Fig. 4A, the current measured in interface may alter the overall response to Kϩ-free solutions.
cells expressing SK3 channels remained active and was insensi- That the whole-cell currents do not fall to zero in Kϩo-free solutions (Fig. 4B) may reflect the presence of this residual Ko .
cells expressing SK4 (Fig. 4B) were sensitive to the presence of The second functional feature we explored was the temper- Kϩo, displaying much less activity in cells preincubated without ature sensitivity of the Gardos channel. Previous work (32) had Kϩo. The cells studied in Fig. 4B were incubated in the absence demonstrated that the open probability (Po) of Gardos channels, o for 3–5 h and were refractory to the addition of Ko after studied in inside-out patches of human erythrocytes, was re- this time (data not shown). We then studied whether the markably sensitive to temperature: the Po at 30°C was Ϸ0.6 expressed SK4 channel could be activated by Kϩo after shorter falling to Ϸ0.1 at 37°C. We examined this effect of temperature incubation times in its absence. Fig. 4C shows the time course of on Gardos channel-mediated Kϩ (Rbϩ) flux in intact human decay in SK4 activity during 2 h incubation in the absence of Kϩ PHYSIOLOGY
erythrocytes as well as the response of the Po values of SK4 As shown in Fig. 4D, the cells can respond to the addition of Kϩo channels expressed in CHO cells to changes in temperature.
PNAS ͉ June 10, 2003 ͉ vol. 100 ͉ no. 12 ͉ 7369
less than those obtained in patched red cell membranes (32), because the Kϩ fluxes were faster at 27 than at 37°C.
Because of the above results, we were interested in evaluating the temperature dependence of Ca2ϩ-activated Kϩ transport through SK4 channels expressed in CHO cells as shown in Fig. 5. Fig. 5 A and B show single-channel activity on two time scales recorded at 25 and 35°C. The results are summarized in the bar graphs (Fig. 5C), where it is clear the Po value at 35°C is less than that at 25°C, consistent with the previous intact red cell flux studies. The observation that the differences in Po values are less than expected based on previous results (32) may indicate that the membrane environment surrounding SK4 channels expressed in CHO cells exhibits substantial differences in the temperature-dependent lipid phase transitions as compared with that present in red cells. The differences may also be due to modulators such as calpromotin (24, 25), which is thought to be required for optimum Gardos activity.
We tested a purified sample of thioredoxin peroxidase that has been shown to be identical to calpromotin (23). The results presented in Fig. 5D indicate that it has no effect on the value of Po compared with the controls (Fig. 5C). In addition, we prepared a hemolysate from red cells according to established procedures (24, 25) that should ensure it contained calpromotin; addition of this lysate to the bathing medium in inside-out patches, as in Fig. 5D, was without The sensitivity of hSK4 channels to changes in temperature. Results of single-channel recordings from inside-out patches excised from CHO cells Discussion
stably expressing hSK4 are presented. The bathing solution contained 1 Mfree Ca2ϩ with 130 mM Kϩ on both sides of the patch (see Methods). Record- The main result of the work reported in this paper is that the ings were performed at Ϫ80 mV first at 25°C and then, within 10 s, at 35°C. The Gardos channel of human red blood cells is coded for by the single-channel activity is shown in A and on an expanded time scale in B. The human SK4 (i.e., KCNN4) gene, as described above in connec- bars labeled 35°C in C and D represent the normalized values at 25°C (taken as tion with Figs. 1–3. This is based first on analysis of RNA from 1.0) of the open probability (Po) of each channel from a given patch. The human reticulocytes and cultured human erythroid progenitor results presented in C represent control characteristics of SK4 channels, cell, in which we found only the message for SK4 and second, on whereas in D, thioredoxin peroxidase (see Methods) has been added to the the use of an antibody directed against SK4. All of these cytoplasmic bathing medium for reasons explained in the text. The error bars preparations were characterized by ‘‘␤ profiling,’’ indicating that are Ϯ SEM where n ϭ 6 in C and n ϭ 4 in D. Lumping the results of C and D contamination from leukocytes and platelets was not detectable.
together, the mean difference between the values at 25 compared with 37°Cis 0.731 Ϯ 0.072 SEM, with P Ͻ 0.05.
Because SK4 is found in human leukocytes (3), this also means that caution must be exercised in interpreting studies (e.g., ref.
7) where embryonic stem cells have been analyzed for SK and The results of net Kϩ efflux experiments in human erythrocytes other isoforms, given that these preparations are likely to be are presented in Table 2. The details of the protocols varied for each contaminated with nonerythroid forms (see, e.g., refs. 35 and experiment (see figure legends and Methods). The primary aim was 36). Additional evidence is provided by Western analysis of to pretreat cells in such a manner that when they were divided and human progenitor cells and erythrocyte ghosts, where it is clearly placed at the two different temperatures, the efflux characteristics shown (Fig. 3) that the SK4 protein is present.
reflected differences in Gardos channel activity and not differences Other support for the conclusion that the SK4 isoform is contents, metabolic states, and possible interfering responsible for the Gardos channel is found in the parallels in membrane transport processes during their subsequent incubation.
function that are displayed in comparisons of human red cells However, this at best is an assumption and is the reason for changing and expressed SK4 channels. This is seen in the effects on the protocols. When the cells contained ClϪ as the principal anion, Ca2ϩ-activated Kϩ channels of preincubation of cells in the i , regardless of protocol design, was the same at 27 presence and absence of Ko (Fig. 4) and in the decrease in Po and 37°C (data not shown), similar to previous results (33). For of channels when the temperature is raised from 25–27°C instance, net Kϩ effluxes were also carried out as part of the same to 35–37°C (Table 2 and Fig. 5). It should be emphasized, experiment (B in Table 2) but where ClϪ replaced SCNϪ in the however, that neither the parallel in the effects of Kϩo nor the protocol. The comparable efflux rate constants (hrϪ1) at 27 and temperature can be considered definitive, because their relative 37°C were 0.33 and 0.35, respectively. Because we thought that effects fall short of expectations based on intact red cell͞ghost the Kϩ efflux was rate limited by the membrane’s permeability to results (9, 31) or determination of Po by patch analyses of red ClϪ, i.e., PK͞PCl ϾϾ 1, we switched the principal anion to SCNϪ be- cell membranes (32). On the other hand, previous observations cause it has been shown that, under the conditions we were using, by others, as referred to in the Introduction, have defined critical PSCN ϾϾ PK (34). In the results shown in Table 2, the efflux of Kϩ, biophysical and pharmacological properties that implicate the SK4 isoform as being the Gardos channel in human red than at 37°C. The variation in the flux values seen in the different cells. Thus the single-channel conductance of heterologously experiments is due primarily to the different protocols used. An expressed SK4 channels as well as their electrical characteristics important caveat in these experiments is that there may be heter- and pharmacological profiles were essentially the same as ogeneity in the response of the cell population to Gardos channel that seen for Gardos channels in intact human red cells (7, 9, 10, activity, because the extrapolated equilibrium end points varied and 13, 32). It is possible that the Gardos channel may have one or were not necessarily the same for the two temperatures in most more modulators, still to be defined, that are necessary for the experiments (data not shown). Even so, the temperature depen- hSK4 isoform to display fully the known characteristics of the dence of Kϩ efflux (Table 2) in intact red cells appears to reflect Gardos channel in human red blood cells. Candidates for such changes in the Po of the Gardos channel that are consistent with but modulators, in addition to the lipid environment, may be cal- 7370 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.1232342100
modulin (13, 30, 37, 38), redox systems (39, 40), and͞or protein invaluable advice and help during the course of this work. In addition, we thank Drs. G. Moss for the rSK3 construct and B. Kristensen for the sample of thioredoxin peroxidase. This work was supported in part by We thank Drs. M. Caplan, C. Canessa, V. Lew, K. Chandler, S.
National Institutes of Health Grant HL09906 (to J.F.H.) and by a grant Basavappa, V. Rajendran, P. Bennekou, and P. Christophersen for from the Wendy Will Case Cancer Fund (to A.W.).
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PNAS ͉ June 10, 2003 ͉ vol. 100 ͉ no. 12 ͉ 7371
Fachinformation Name des Präparates Hippurat-lod-123 Heider Injektionslösung Zusammensetzung Wirkstoff: Hilfstoffe: Natri citras, Natri phosphates, Aqua q.s. ad solutionem Galenische Form und Wirkstoffmenge pro Einheit Sterile, klare, wässrige Lösung. Wirkstoffmenge: 18,5 – 370 MBq Indikation • Untersuchungen der Nierenfunktion mittels Gammakamera Quantit