The SK3 Subunit of Small Conductance Ca2+-activated K+ Channels Interacts with Both SK1 and SK2 Subunits in a Heterologous Expression System
2004; Elsevier BV; Volume: 279; Issue: 2 Linguagem: Inglês
10.1074/jbc.m308070200
ISSN1083-351X
AutoresAlan Monaghan, David Benton, Parmvir K. Bahia, Ramine Hosseini, Yousaf A. Shah, D.G. Haylett, Guy W. J. Moss,
Tópico(s)Neuroscience and Neuropharmacology Research
ResumoThe aim of this study was to determine whether functional heteromeric channels can be formed by co-assembly of rat SK3 (rSK3) potassium channel subunits with either SK1 or SK2 subunits. First, to determine whether rSK3 could co-assemble with rSK2 we created rSK3VK (an SK3 mutant insensitive to block by UCL 1848). When rSK3VK was co-expressed with rSK2 the resulting currents had an intermediate sensitivity to UCL 1848 (IC50 of ∼5nm compared with 120 pm for rSK2 and >300 nm for rSK3VK), suggesting that rSK3 and rSK2 can form functional heteromeric channels. To detect co-assembly of SK3 with SK1, we initially used a dominant negative construct of the human SK1 subunit (hSK1YP). hSK1YP dramatically reduced the SK3 current, supporting the idea that SK3 and SK1 subunits also interact. To determine whether these assemblies were functional we created rSK3VF, an rSK3 mutant with an enhanced affinity for tetraethylammonium chloride (TEA) (IC50 of 0.3 mm). Co-transfection of rSK3VF and hSK1 produced currents with a sensitivity to TEA not different from that of hSK1 alone (IC50 ∼15 mm). These results suggest that hSK1 does not produce functional cell-surface assemblies with SK3. Antibody-staining experiments suggested that hSK1 may reduce the number of functional SK3 subunits reaching the cell surface. Additional experiments showed that co-expression of the rat SK1 gene with SK3 also dramatically suppressed SK current. The pharmacology of the residual current was consistent with that of homomeric SK3 assemblies. These results demonstrate interactions that cause changes in protein trafficking, cell surface expression, and channel pharmacology and strongly suggest heteromeric assembly of SK3 with the other SK channel subunits. The aim of this study was to determine whether functional heteromeric channels can be formed by co-assembly of rat SK3 (rSK3) potassium channel subunits with either SK1 or SK2 subunits. First, to determine whether rSK3 could co-assemble with rSK2 we created rSK3VK (an SK3 mutant insensitive to block by UCL 1848). When rSK3VK was co-expressed with rSK2 the resulting currents had an intermediate sensitivity to UCL 1848 (IC50 of ∼5nm compared with 120 pm for rSK2 and >300 nm for rSK3VK), suggesting that rSK3 and rSK2 can form functional heteromeric channels. To detect co-assembly of SK3 with SK1, we initially used a dominant negative construct of the human SK1 subunit (hSK1YP). hSK1YP dramatically reduced the SK3 current, supporting the idea that SK3 and SK1 subunits also interact. To determine whether these assemblies were functional we created rSK3VF, an rSK3 mutant with an enhanced affinity for tetraethylammonium chloride (TEA) (IC50 of 0.3 mm). Co-transfection of rSK3VF and hSK1 produced currents with a sensitivity to TEA not different from that of hSK1 alone (IC50 ∼15 mm). These results suggest that hSK1 does not produce functional cell-surface assemblies with SK3. Antibody-staining experiments suggested that hSK1 may reduce the number of functional SK3 subunits reaching the cell surface. Additional experiments showed that co-expression of the rat SK1 gene with SK3 also dramatically suppressed SK current. The pharmacology of the residual current was consistent with that of homomeric SK3 assemblies. These results demonstrate interactions that cause changes in protein trafficking, cell surface expression, and channel pharmacology and strongly suggest heteromeric assembly of SK3 with the other SK channel subunits. Small conductance Ca2+-activated potassium channels (SK 1The abbreviations used are: SK, small conductance calcium-activated potassium; PBS, phosphate-buffered saline; ABS, antibody blocking solution; GFP, green fluorescent protein; TEA, tetraethylammonium chloride. channels) are widely expressed throughout the central and peripheral nervous systems. In many neurons SK channels underlie some components of the post-spike after-hyperpolarization (see e.g. Ref. 1.Sah P. Faber E.S. Prog. Neurobiol. 2002; 66: 345-353Crossref PubMed Scopus (413) Google Scholar). They also have important functions in non-neuronal tissues. Native SK channels have a characteristic pharmacology. They can be blocked by the bee venom toxin apamin and several selective small molecule blockers that we have developed (such as UCL 1848) that are active at nanomolar or subnanomolar concentrations (2.Chen J.Q. Galanakis D. Ganellin C.R. Dunn P.M. Jenkinson D.H. J. Med. Chem. 2000; 43: 3478-3481Crossref PubMed Scopus (54) Google Scholar, 3.Benton D.C.H. Dunn P.M. Chen J.Q. Galanakis D. Ganellin C.R. Malik-Hall M. Shah M. Haylett D.G. Jenkinson D.H. Br. J. Pharmacol. 1999; 128: 39PGoogle Scholar, 4.Faber E.S. Sah P. J. Neurosci. 2002; 22: 1618-1628Crossref PubMed Google Scholar). Molecular cloning studies have identified three closely related genes (SK1, -2, and -3) which code for SK channel subunits in mammalian cells (5.Kohler M. Hirschberg B. Bond C.T. Kinzie J.M. Marrion N.V. Maylie J. Adelman J.P. Science. 1996; 273: 1709-1714Crossref PubMed Scopus (804) Google Scholar, 6.Joiner W.J. Wang L.Y. Tang M.D. Kaczmarek L.K. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 11013-11018Crossref PubMed Scopus (318) Google Scholar, 7.Chandy K.G. Fantino E. Wittekindt O. Kalman K. Tong L.L. Ho T.H. Gutman G.A. Crocq M.A. Ganguli R. Nimgaonkar V. Morris-Rosendahl D.J. Gargus J.J. Mol. Psychiatry. 1998; 3: 32-37Crossref PubMed Scopus (184) Google Scholar). In both Xenopus oocytes and mammalian cell lines, expression of the rat homologues of SK2 and SK3 (rSK2 and rSK3 respectively) results in the formation of functional homomeric SK channels. Further, both homomeric rSK2 and homomeric rSK3 channels can be blocked by either apamin or UCL 1848 at concentrations that are similar to those reported for native channels (8.Hosseini R. Benton D.C.H. Dunn P.M. Jenkinson D.H. Moss G.W.J. J. Physiol. 2001; 535: 323-334Crossref PubMed Scopus (64) Google Scholar). The potencies of both compounds depend on the subunit composition of the channel, with the IC50 for blocking homomeric SK2 channels being ∼18-fold lower than for SK3. The behavior of SK1 is different to that of other SK genes. Initial expression studies of the human SK1 gene (hSK1) showed that it can produce functional channels in the Xenopus oocyte expression system and these channels are insensitive to apamin at concentrations of up to 100 nm (5.Kohler M. Hirschberg B. Bond C.T. Kinzie J.M. Marrion N.V. Maylie J. Adelman J.P. Science. 1996; 273: 1709-1714Crossref PubMed Scopus (804) Google Scholar). Subsequent work, however, expressing hSK1 in mammalian cell lines, showed that most cells produce channels that are blocked by apamin with an IC50 of ∼3–12 nm. A few cells also produce an apamin-insensitive current component (9.Shah M. Haylett D.G. Br. J. Pharmacol. 2000; 129: 627-630Crossref PubMed Scopus (101) Google Scholar, 10.Strobaek D. Jorgensen T.D. Christophersen P. Ahring P.K. Olesen S.P. Br. J. Pharmacol. 2000; 129: 991-999Crossref PubMed Scopus (162) Google Scholar). More recent oocyte work suggests that, in fact, expression of the human SK1 gene also produces some apamin-sensitive channels in this system (11.Grunnet M. Jensen B.S. Olesen S.P. Klaerke D.A. Pflugers Arch. 2001; 441: 544-550Crossref PubMed Scopus (115) Google Scholar). The reason for this behavior is unclear and it consequently remains unknown whether SK1 forms apamin-sensitive channels or apamin-insensitive channels, or both, in vivo. Interestingly, the rat SK1 gene differs from its human counterpart because it does not produce functional channels when expressed alone in either oocytes or mammalian cell lines (12.Bowden S.E. Fletcher S. Loane D.J. Marrion N.V. J. Neurosci. 2001; 21: RC175Crossref PubMed Google Scholar). The overlapping patterns of expression for SK1, SK2 and SK3 within the CNS (5.Kohler M. Hirschberg B. Bond C.T. Kinzie J.M. Marrion N.V. Maylie J. Adelman J.P. Science. 1996; 273: 1709-1714Crossref PubMed Scopus (804) Google Scholar, 13.Stocker M. Krause M. Pedarzani P. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 4662-4667Crossref PubMed Scopus (336) Google Scholar, 14.Stocker M. Pedarzani P. Mol. Cell Neurosci. 2000; 15: 476-493Crossref PubMed Scopus (316) Google Scholar) raise the possibility that heteromeric assembly of SK subunits may occur. That co-assembly may occur is also suggested by the work of Ishii et al. (15.Ishii T.M. Maylie J. Adelman J.P. J. Biol. Chem. 1997; 272: 23195-23200Abstract Full Text Full Text PDF PubMed Scopus (230) Google Scholar) where it was shown that injection of an SK1-SK2 dimer or co-injection of mRNA for hSK1 and rSK2 into Xenopus oocytes resulted in currents with an apamin sensitivity between that of homomeric hSK1 and rSK2. Following this observation we have recently shown that co-transfection of the rat SK1 and SK2 genes in HEK cells produces channels with a novel pharmacology, suggesting that subunits from these genes can also assemble to form functional heteromeric channels (16.Benton D.C.H. Monaghan A.S. Hosseini R. Bahia P.K. Shah Y. Haylett D.G. Moss G.W.J. J. Physiol. 2003; 553: 13-19Crossref PubMed Scopus (62) Google Scholar). Despite the progress being made toward understanding SK1/SK2 interactions, much less is known of the interactions between SK3 and the other SK subunits. Interestingly, however, it has been reported that a fragment of SK3, when transfected into the Jurkat cell line, acts as a dominant negative, suppressing the endogenously expressed SK2 current (17.Miller M.J. Rauer H. Tomita H. Rauer H. Gargus J.J. Gutman G.A. Cahalan M.D. Chandy K.G. J. Biol. Chem. 2001; 276: 27753-27756Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar). This finding suggests that SK2 and SK3 may be able to form a heteromeric complex. The principle aim of the present work was to examine, in more detail, the possibility that SK3 interacts with the other SK channel subunits; SK1 and SK2. Constructs—The rat SK1, SK2, and human IK1 (SK4) genes subcloned into the pTracer or pcDNA3 mammalian expression vectors were a generous gift from Prof. Len Kaczmarek and Dr. William Joiner (Yale University). The rat SK1 clone (accession number AF000973) encodes a full-length transcript and was re-engineered to eliminate the 5′-untranslated region and replace it with an optimal Kozak sequence (gccacc) just prior to the start methionine (starting protein sequence MSSRSH...). The rat SK3 gene (accession number AF292389) was previously cloned from a rat SCG library (8.Hosseini R. Benton D.C.H. Dunn P.M. Jenkinson D.H. Moss G.W.J. J. Physiol. 2001; 535: 323-334Crossref PubMed Scopus (64) Google Scholar) and subcloned into the pcDNA 3.1 Zeo+ plasmid (Invitrogen). The human hSK1 clone (accession number U69883) was a generous gift from Prof. J. P. Adelman (Vollum Inst.). Mutations were made using the QuikChange Site-directed Mutagenesis kit (Stratagene) or by standard overlap extension PCR. Constructs were sequenced on an ABI 377 sequencer using the Big Dye II sequencing kit. The TEA-sensitive mutations, which substitute phenylalanine for valine in rSK2 (SK2VF) and rSK3 (SK3VF) are at structurally equivalent amino acid positions (366 and 515 in rSK2 and rSK3, respectively). The UCL 1848-insensitive mutation of rSK3 (SK3VK) substitutes a lysine residue for a valine at position 491. In the dominant negative construct of hSK1 (hSK1YP) proline is substituted for tyrosine at position 351. Plasmid DNA for transfection was purified using Maxi Prep or Midi Prep kits (Qiagen). Maintenance and Transient Transfection of HEK 293 Cells—HEK 293 cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% v/v fetal calf serum, 2 mm l-glutamine, penicillin (100 units/ml), and streptomycin (100 μg/ml). Cells were plated into 35-mm culture dishes and transfected using LipofectAMINE 2000 (Invitrogen). The protocol for transfection of HEK cells was varied slightly according to the precise aims of the experiment. For studies of the pharmacology of homomeric SK channels we used 1 μg of plasmid. For experiments where hSK1YP or rSK1 were co-expressed with another SK gene we used 2 μg of hSK1YP or rSK1 construct to 1 μg of the other. For co-expression of hSK1 and the change-of-function mutants 1 μg of each was used. For immunohistochemistry cells were transfected with 1 μg of rSK3 alone or with 1 μg of rSK3 together with 4 μg of hSK1 or rSK1. In addition, for all experiments, cells were co-transfected with 1 μg of QBI plasmid DNA (Qbiogene), which expresses GFP, allowing identification of transfected cells. LipofectAMINE 2000 (1 μl/μg of DNA) and plasmid DNA were mixed in Opti-MEM and added to cells plated into 35-mm culture dishes. After incubation overnight the cells were re-plated into 35-mm dishes and used within 2 days for recording. Cells used for immunohistochemistry were plated on 18-mm square glass coverslips. Immunohistochemistry—Transfected cells plated on glass cover slips were washed three times in phosphate-buffered saline (PBS) before being fixed for 10 min using freshly made 4% w/v paraformaldehyde (in PBS). Cells were washed in PBS again and permeabilized using 100% methanol for 10 min. After a further wash in PBS the cells were then left in an antibody-blocking solution (ABS) consisting of 2% w/v bovine serum albumin and 2% w/v horse serum in PBS for 1 h. The cells were then incubated for 4 h in the primary anti-rSK3 antibody (Chemicon) at a concentration of 0.3 μg/μl. To remove excess primary antibody, the cells were washed three times in PBS containing 0.1% v/v Tween-20 and then incubated in a 1:200 dilution of a Cy3-conjugated goat anti-rabbit secondary antibody (Chemicon) for 1 h. All antibodies were diluted in ABS. Following incubation, cells underwent a final wash in PBS with 0.1% v/v Tween-20. Coverslips were mounted onto slides (previously cleaned with ethanol) using a small drop of antifade mount (Vector Laboratories Inc.). All staining operations were carried out at room temperature (∼22 °C). Stained cells were viewed with a Leica TCS confocal microscope. Electrophysiology—Currents were recorded from HEK 293 cells using conventional whole cell voltage clamp methods. The bathing solution contained (in mm): NaCl 140, KCl 5, MgCl2 1, CaCl2 2, glucose 10, HEPES 10, and the pH was adjusted to 7.4 with NaOH. The pipette filling solution contained (in mm): KCl 130, HEPES 10, K2HEDTA 5, and CaCl2 1.2 (free Ca2+ 1 μm). The pH was adjusted to 7.2 with KOH. The free Ca2+ concentration was calculated using stability constants from Martell and Smith (18.Martell A. Smith R. Critical Stability Constants. Plenum Press, New York1974: 199Google Scholar). The 50 μm (unbuffered) calcium pipette filling solution contained (in mm): KCl 140, HEPES 10, and 0.05 CaCl2 (pH adjusted to 7.2 with KOH). Patch pipettes were fabricated from 1.5 mm o.d. borosilicate glass (Harvard), fire-polished, and coated with Sylgard resin. They had resistances of 2–4 MΩ when filled with the above solution. Experiments were conducted at room temperature (20–25 °C). Membrane currents were recorded with either a List EPC7 amplifier using a Digidata 1320A interface and pClamp 8.2 software (Axon Instruments) for acquisition, or a HEKA EPC9 patch clamp amplifier under control of Pulse software. Data were filtered at 1 kHz and digitized at 5 kHz. Acquired current traces were analyzed with either Clampfit 8.2 or HEKA Pulsefit. Routinely, cells were held at –80 mV and current-voltage relationships generated by applying 100 ms voltage steps to potentials between –120 mV and +40 mV. Since untransfected (wild-type) HEK 293 cells exhibit an outwardly rectifying current at potentials positive to 0 mV the effect of blockers was measured at –20 mV. Similarly, comparisons between currents in cells transfected with or without dominant negative construct were made at this potential. Data Analysis—The effect of blocking agents was expressed as the current at –20 mV in the presence of blocker as a percentage of that in its absence. The resulting concentration-inhibition curves were fitted by the Hill equation in the form of Equation 1, y100=IC50nH[I]nH+IC50nH(Eq. 1) where y is the current in the presence of blocker as a percentage of the control, [I] is the concentration of inhibitor, nH is the Hill coefficient, and IC50 is the concentration of blocker that reduces the current to 50% of the control value. Curve fitting was performed by the method of least squares using the curve fitting routine of Origin 6.1 (Microcal). Comparison of the currents developed in the presence and almost complete absence of free intracellular Ca2+ suggested that under the conditions used virtually all (>95%) of the current was due to activation of SK channels (data not shown). The curves were, therefore, fitted assuming that complete inhibition of the current was possible. Where appropriate, values are quoted as the mean ± S.E. The Students t test (two-tailed, unpaired) was used to determine the significance of results. Drugs and Reagents—All materials used for tissue culture were obtained from Invitrogen. UCL 1848 (8,14-diaza-1,7-(1, 4)-diquinolinacyclotetradecaphanedium tetratrifluoroacetate) was synthesized in the Department of Chemistry, UCL as previously described (2.Chen J.Q. Galanakis D. Ganellin C.R. Dunn P.M. Jenkinson D.H. J. Med. Chem. 2000; 43: 3478-3481Crossref PubMed Scopus (54) Google Scholar). Tetraethylammonium chloride (TEA) was purchased from Sigma. HEPES and HEDTA were from Calbiochem. All other reagents were of Analar quality and obtained from Merck or Sigma. SK3 Can Co-assemble With SK2 Forming Functional Channels—It has previously been reported that expression of a dominant negative of SK3 can suppress SK2 currents in Jurkat cells (17.Miller M.J. Rauer H. Tomita H. Rauer H. Gargus J.J. Gutman G.A. Cahalan M.D. Chandy K.G. J. Biol. Chem. 2001; 276: 27753-27756Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar), suggesting that SK3 and SK2 subunits co-assemble in this expression system. However, it is not yet clear whether functional channels are made by co-assembly of SK2 and SK3. We addressed this question using a mutant of SK3, SK3VK, which is very insensitive to block by apamin 2G. W. J. Moss, A. S. Monaghan, and Y. A. Shah, unpublished observations. and UCL 1848, a selective small molecule blocker developed in our laboratories (2.Chen J.Q. Galanakis D. Ganellin C.R. Dunn P.M. Jenkinson D.H. J. Med. Chem. 2000; 43: 3478-3481Crossref PubMed Scopus (54) Google Scholar, 3.Benton D.C.H. Dunn P.M. Chen J.Q. Galanakis D. Ganellin C.R. Malik-Hall M. Shah M. Haylett D.G. Jenkinson D.H. Br. J. Pharmacol. 1999; 128: 39PGoogle Scholar). In control experiments on cells expressing SK3VK alone, 300 nm UCL 1848 had little or no effect on the amplitude of currents (Fig. 1, B and D). The advantage of using UCL 1848 as a blocker (rather than apamin) is that it has a similar potency for blocking SK2 to apamin but, unlike apamin, its action on SK2 is much more rapidly reversed, so we were able to obtain full recovery after ∼3 min washout (Fig. 1, A and C). This cannot be obtained with apamin (10.Strobaek D. Jorgensen T.D. Christophersen P. Ahring P.K. Olesen S.P. Br. J. Pharmacol. 2000; 129: 991-999Crossref PubMed Scopus (162) Google Scholar). Cells expressing SK2 alone were blocked by UCL 1848 with an IC50 of 110 ± 26 pm (Fig. 1D), an essentially identical value to the one previously reported (8.Hosseini R. Benton D.C.H. Dunn P.M. Jenkinson D.H. Moss G.W.J. J. Physiol. 2001; 535: 323-334Crossref PubMed Scopus (64) Google Scholar). However, when rSK3VK was co-expressed with SK2 the concentration-inhibition curve was shifted to lie between those of SK2 and SK3VK (Fig. 1, C and D). The intermediate sensitivity of the channels formed is particularly obvious in recordings that were sufficiently long and stable to apply multiple doses of drug; one such experiment is shown in Fig. 1C. Clearly the majority of current is mediated by SK3VK-SK2 heteromeric channels in these co-transfected cells or the second application of drug would not provide such a large increase in the fraction of channels blocked. Our results are thus consistent with the idea that SK3, like SK1, can assemble with SK2 forming functional heteromeric channels in vitro. Effect of a Dominant Negative hSK1 Subunit on rSK3 Currents—Next, to examine possible assembly of SK3 with SK1 we used a dominant negative construct of hSK1 (hSK1YP) (Fig. 2). We performed three types of control experiments with this construct. First, we tested hSK1YP co-expression with hSK1 to make sure that it can knock down the wild-type SK1 currents. To do this we compared the size of currents in cells transfected using SK1 alone with those transfected using the same quantity of SK1 and a 2-fold excess of hSK1YP. As expected, the hSK1YP construct substantially reduced the SK1 current (Fig. 2D). Second, to confirm the effectiveness of this construct in co-assembly experiments, we expressed SK1YP with the wild-type rSK2 gene. The human SK1 gene and the rat SK2 gene have been shown to form subunits that co-assemble in oocytes. We therefore expected to see knock down of SK2 current. Again we compared the size of currents in cells transfected using SK2 alone with those of cells transfected using the same quantity of SK2 and a 2-fold excess of SK1YP. As expected, there was a substantial reduction in currents when cells were co-transfected with the dominant negative (Fig. 2, B and D). This result is consistent with the idea that in mammalian cells, as well as in oocytes, SK1 and SK2 can co-assemble (even though the pharmacological properties of hSK1 appear to be quite different in the two expression systems). Interestingly, there was more current remaining than would be predicted if both constructs express equally well, subunits assembled at random and only a single subunit of hSK1YP were sufficient to render SK1/SK2 channels nonfunctional. The reason for this is not clear. It may reflect either a partial recovery of function, preferred stoichiometries of the SK1/SK2 interaction or variations in expression levels. However, to ensure that overexpression of a competing plasmid does not itself cause the apparent reduction in SK currents, we performed a final control experiment testing the effect of hSK1YP on the current in cells transfected with hIK1. As in the previous cases, we used a 2-fold excess of the hSK1YP construct over the hIK1 plasmid and compared the currents against those obtained using hIK1 alone. In these experiments the magnitude of the mean hIK1 current was slightly, but not significantly, reduced as a result of co-transfection with hSK1YP (Fig. 2, C and D). This suggests that competition for expression by a 2-fold excess of the hSK1YP construct does not account for the reduced currents seen when hSK1YP is transfected with wild-type SK1 or SK2. The hSK1YP construct thus seems suitable for assessing subunit co-assembly. Having completed these control experiments, we compared cells transfected with SK3 alone or cells transfected with the same quantity of SK3 and a 2-fold excess of dominant negative. The records shown in Fig. 2, A and D demonstrate that SK3 currents were greatly depressed in the presence of hSK1YP. Thus, the effect of hSK1YP on rSK3 currents strongly suggests an interaction occurs between these two channel subunits. However, this result does not prove that hSK1 and rSK3 can co-assemble to form functional channels at the cell surface. In an attempt to detect the presence of functional heteromeric channels we therefore used the change-of-function strategy described in the next section. TEA-sensitive SK3 and SK2 Mutants—Expression of hSK1 in HEK 293 cells produces currents, which are relatively insensitive to block by TEA (Fig. 3A). In our control experiments, TEA blocked homomeric hSK1 channels, rSK2 channels, or rSK3 channels, with IC50 values of 14.1 ± 1.0, 2.8 ± 0.7, and 8.7 ± 1.8 mm respectively (Fig. 3D, data not shown and Fig. 6). The value for hSK1 is similar to that reported from oocyte experiments (15.Ishii T.M. Maylie J. Adelman J.P. J. Biol. Chem. 1997; 272: 23195-23200Abstract Full Text Full Text PDF PubMed Scopus (230) Google Scholar). The TEA sensitivity of K+ channels has been shown to be altered by changing just a single amino acid in the potassium channel pore sequence; at the right position, an aromatic residue provides sub-millimolar TEA affinity (19.Heginbotham L. MacKinnon R. Neuron. 1992; 8: 483-491Abstract Full Text PDF PubMed Scopus (377) Google Scholar). We therefore mutated SK3 to introduce a phenylalanine at this position creating the mutant rSK3VF. This mutation caused the expected increase in sensitivity to TEA; the concentration-inhibition curve was fitted to a single component Hill-Langmuir equation with an IC50 for block by TEA of 0.31 ± 0.07 mm and an nH value of 0.9 ± 0.2 (Fig. 3, B and D). Surprisingly, when rSK3VF was co-expressed with hSK1 the concentration-inhibition curve for TEA was virtually identical to that of homomeric hSK1 (IC50 16 ± 1.5 mm, nH 0.8 ± 0.1, Fig. 3, C and D). This suggests that SK3VF made little or no contribution to the observed current.Fig. 6Co-expression of rSK1 and rSK3VF. TEA concentration-inhibition curves for wild-type rSK3 (▴) and rSK3VF alone (•) or co-expressed with rSK1 (□). The Hill equation fits the data with IC50 values of 8.6 ± 1.6, 0.31 ± 0.07, and 0.24 ± 0.02 mm, respectively. Each data point is the mean of three or more experiments.View Large Image Figure ViewerDownload Hi-res image Download (PPT) To validate the approach used above we repeated the experiment by creating rSK2VF, an equivalent mutation in the SK2 channel. We would expect to be able to obtain a dose response curve between that of SK1 and SK2VF when these subunits are co-expressed because assembly of SK2 with hSK1 has been previously described (15.Ishii T.M. Maylie J. Adelman J.P. J. Biol. Chem. 1997; 272: 23195-23200Abstract Full Text Full Text PDF PubMed Scopus (230) Google Scholar). It would also be expected from our dominant negative studies. In control experiments, SK2VF, like SK3VF, displays a marked increase in sensitivity to TEA; the IC50 for block being 0.31 ± 0.05 mm, nH 1.0 ± 0.2 (Fig. 4, A and C). An additional observation was that introduction of phenylalanine at this site changed the time course of the current seen when a voltage jump was applied (Fig 4A). (There is also a suggestion of this same effect in some records obtained with our SK3VF mutant (Fig. 3), but it is less pronounced.) The process underlying this change is as yet unknown and merits further study. In the present case, however, we noted that cells transfected with SK2VF and hSK1 tended to produce SK current with kinetics that were intermediate between those of homomeric SK2VF and homomeric hSK1 (Fig. 4B). Further, in co-transfected cells it was possible to determine that current sensitivity to TEA was between that of hSK1 and rSK2VF, as shown in Fig. 4B and by the averaged data shown in Fig. 4C. The concentration-inhibition curve for co-transfected cells has an IC50 of 3.5 ± 0.9 mm. In addition to a change in the IC50, if co-assembly is occurring, then one might expect the dose-response curve to become rather shallow because it would represent a number of different populations where various stoichiometries of hSK1 and rSK2VF subunits have formed. When, for the purpose of comparison, both sets of data are fitted to a single component Hill curve, the co-expressed channel data is clearly best fitted with a smaller Hill coefficient for TEA block (nH value of 0.55 ± 0.10, compared with values of 1.2 ± 0.1 and 1.0 ± 0.2 for hSK1 and rSK2VF homomers, respectively). Again this argues in favor of co-assembly. Thus, the interaction of SK2 with SK1 in mammalian cells is entirely consistent with the idea that heteromeric channel assemblies can be formed. In contrast, our studies of the interaction between SK3 and SK1 do not provide evidence of functional heteromeric assemblies. Co-expression of rSK3 and rSK1—We became concerned that the suppression of rSK3 by hSK1 could be an artifact of using the human SK1 clone in combination with rat SK3. We therefore decided to examine currents arising from expression of the rat SK1 gene with rSK3. We started by comparing Ca2+-activated K+ currents in HEK 293 cells transfected with rSK3 either alone or in combination with rSK1. As expected, cells transfected with rSK3 alone exhibited large currents with an approximately linear current-voltage relationship, which reversed close to the predicted value of Ek (Fig. 5, A and D). When cells were transfected with SK3 together with rSK1, however, currents were markedly reduced (Fig. 5, A and C) although, the reversal potential was unchanged (Fig. 5D) and a small SK-like current was still clearly present. This result appears to confirm an interaction between SK1 and SK3 that reduces the SK3 current component, as was seen with the human SK1. However, a small SK current might be explained if SK1 and SK3 co-assemble to form a channel that is less sensitive to Ca2+ activation. In order to test this possibility we repeated the experiment, this time recording with a pipette solution containing 50 μm Ca2+. As shown in Fig. 5, B and C the results obtained were virtually identical to those with 1 μm free Ca2+. Thus, the reduction in current does not reflect a shift in the Ca2+ activation curve. These results might still be explained in a number of ways. It could be, for example, that this residual current is carried by SK1/SK3 channels, which have a smaller conductance or lower open probability than SK3 homotetramers. Alternatively, the remaining currents may reflect a smaller residual SK3 component. In this case, both rSK1 and hS
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