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The Hsk4 (KCNN4) Isoform Is the Ca -Activated K Channel (Gardos

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The Hsk4 (KCNN4) Isoform Is the Ca -Activated K Channel (Gardos The 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 of Rochester 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 Ko and remains refractory to Ko added after exposure to in Chinese hamster ovary (CHO) cells, displays functional 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, human red blood cells. the open probability (Po) of the Gardos channel is markedly Methods 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 cells expressing SK4 (but not SK3). Thus the envelope of evidence cytes and human erythroid progenitor cells purified and cultured indicates that SK4 is the gene that codes for the Gardos channel in as described previously (20). All of these preparations were found to be free of leukocyte and platelet contamination based human red blood cells. This channel is important pathophysiologi- ␤ cally, because it represents the major pathway for cell shrinkage on the criterion of ‘‘ profiling,’’ as previously established (20). 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 our isoforms (SK1–4) of the small conductance Ca2ϩ- ϩ not SK4. To confirm that primers were capable of amplifying a Factivated K 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 dpm͞␮g. 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) (CLONTECH). 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 (mSK4 IKCa) 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 Abbreviations: CLT, clotrimazole; CHO, Chinese hamster ovary. 3 channels are inhibited by apamin (14–17) but not by CLT (18). †To whom correspondence should be addressed at: Department of Cellular and Molecular Obviously, it is necessary to go beyond the foregoing correlations Physiology, Yale University School of Medicine, 333 Cedar Street, New Haven, CT 06520- of channel characteristics to establish which isoform(s) of the SK 8026. E-mail: [email protected]. 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, ϩ and isoform composition of the endogenously expressed Na New Haven, CT 06510. 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 Downloaded by guest on September 29, 2021 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 Specific Expected 7.2). This solution was supplemented with 4.27 or 4.74 mM CaCl2 2ϩ ␮ primer size, bp Primer sequence to achieve free Ca concentrations of 1.0 or 3.0 M, respec- tively. All whole-cell recordings were performed by using 200-ms SK4 1,767 5Ј-GCTGACGACCTGCAAGCCACAGTGG-3Ј ramps from Ϫ120 to ϩ80 mV from a holding potential of Ϫ70 5Ј-GGTAACTGAAGCCTCCCAGAATCCTGG-3Ј mV. Current densities were measured for each cell by dividing 529 5Ј-CCTTTCAGACACACTTTGGCTGATCC-3Ј the current amplitude at 60 mV by the capacitance. 5Ј-CAGTGCTAAGCAGCTCAGTCAGG-3Ј For inside-out patch recordings of stably transfected cells, SK3 1,275 5Ј-GCTCCATCACCCTAATGCCACC-3Ј pipettes were filled with (in mM): 30 KCl, 100 K-gluconate, 1 Ј Ј 5 -GCTGCCAATCTGCTTCTCCAGG-3 MgCl2, and 10 Hepes (pH 7.2), and the cytoplasmic side of the 2,226 5Ј-GCTTGTGCTGCAGGGAATACAGC-3Ј membrane was perfused with (in mM): 30 KCl, 100 K-gluconate, Ј Ј ␮ 2ϩ 5 -GCAACTGCTTGAACTTGTGTACG-3 5 EGTA, 10 Hepes (pH 7.2), and 4.27 CaCl2 (1 M free Ca ). SK2 670 5Ј-CTCCATTGGTTATGGTGACATGGTACC-3Ј In some experiments, lyophilized thioredoxin peroxidase (23), 5Ј-CTAGCTACTCTCTGATGAAGTTGGTGG-3Ј i.e., calpromotin (24, 25), was dissolved in the latter solution at 506 5Ј-GTAGTGGCAAGGAAGCTAGAACTTACC-3Ј a concentration of 10 ␮M and used to perfuse excised inside-out 5Ј-GACCGGGACCGCTCAGCATTG-3Ј patches. All patch recordings were made over a period of 30–60 SK1 1,428 5Ј-CAGGTAGTCATGAACAGCCACAGC-3Ј s at a holding potential of Ϫ80 mV, first at 25°C and subse- 5Ј-GAGCGTGCAGCTCCGATACAAGG-3Ј quently, after rapidly switching (Ͻ10 s), at a controlled bath Ϸ150 5Ј-GGTAGTCATGAACAGCCACAGC-3Ј temperature of 35°C in a temperature-controlled 35-mm tissue 5Ј-CCTGTAGAGAGGCACCCAGC-3Ј culture cup. Electrical heating was mediated via a Pt͞Ir oxide SK1–3 641 5Ј-GATGACTGGMGNATMGCCATGAC-3Ј film on the outside of the cup (Bioptechs, Butler, PA). 5Ј-GGTSAGCTGAGTRTCCATCATG-3Ј .Net K؉ and 86Rb؉ Fluxes In the case of the SK1–3 (i.e., SK1, SK2, and SK3) primer set, M ϭ AorC;Rϭ o Blood was drawn into heparin from normal volunteers and used without delay. Net Kϩ effluxes were AorG;andNϭ A, C, G, or T. ϩ measured by incubating red cells in a low Ko medium for various time periods.
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