The Selectivity Filter May Act as the Agonist-activated Gate in the G Protein-activated Kir3.1/Kir3.4 K+ Channel
2003; Elsevier BV; Volume: 278; Issue: 50 Linguagem: Inglês
10.1074/jbc.m308183200
ISSN1083-351X
AutoresTom W. Claydon, Samy Makary, Katharine M. Dibb, Mark R. Boyett,
Tópico(s)Nicotinic Acetylcholine Receptors Study
ResumoThe Kir3.1/Kir3.4 channel is activated by Gβγ subunits released on binding of acetylcholine to the M2 muscarinic receptor. A mechanism of channel opening, similar to that for the KcsA and Shaker K+ channels, has been suggested that involves translocation of pore lining transmembrane helices and the opening of an intracellular gate at the "bundle crossing" region. However, in the present study, we show that an extracellular gate at the selectivity filter is critical for agonist activation of the Kir3.1/Kir3.4 channel. Increasing the flexibility of the selectivity filter, by disrupting a salt bridge that lies directly behind the filter, abolished both selectivity for K+ and agonist activation of the channel. Other mutations within the filter that altered selectivity also altered agonist activation. In contrast, mutations within the filter that did not affect selectivity had little if any effect on agonist activation. Interestingly, mutation of bulky side chain phenylalanine residues at the bundle crossing also altered both agonist activation and selectivity. These results demonstrate a significant correlation between agonist activation and selectivity, which is determined by the selectivity filter, and suggests, therefore, that the selectivity filter may act as the agonist-activated gate in the Kir3.1/Kir3.4 channel. The Kir3.1/Kir3.4 channel is activated by Gβγ subunits released on binding of acetylcholine to the M2 muscarinic receptor. A mechanism of channel opening, similar to that for the KcsA and Shaker K+ channels, has been suggested that involves translocation of pore lining transmembrane helices and the opening of an intracellular gate at the "bundle crossing" region. However, in the present study, we show that an extracellular gate at the selectivity filter is critical for agonist activation of the Kir3.1/Kir3.4 channel. Increasing the flexibility of the selectivity filter, by disrupting a salt bridge that lies directly behind the filter, abolished both selectivity for K+ and agonist activation of the channel. Other mutations within the filter that altered selectivity also altered agonist activation. In contrast, mutations within the filter that did not affect selectivity had little if any effect on agonist activation. Interestingly, mutation of bulky side chain phenylalanine residues at the bundle crossing also altered both agonist activation and selectivity. These results demonstrate a significant correlation between agonist activation and selectivity, which is determined by the selectivity filter, and suggests, therefore, that the selectivity filter may act as the agonist-activated gate in the Kir3.1/Kir3.4 channel. K+ channels open and close in response to a number of stimuli including voltage or intracellular ligands such as G proteins. The position of the gate or gates that control channel opening and closing remains a matter of interest. Studies on the proton-gated KcsA K+ channel (1.Perozo E. Cortes D.M. Cuello L.G. Science. 1999; 285: 73-78Crossref PubMed Scopus (496) Google Scholar, 2.Liu Y.S. Sompornpisut P. Perozo E. Nat. Struct. Biol. 2001; 8: 883-887Crossref PubMed Scopus (185) Google Scholar), the voltage-gated Shaker K+ channel (3.Liu Y. Holmgren M. Jurman M.E. Yellen G. Neuron. 1997; 19: 175-184Abstract Full Text Full Text PDF PubMed Scopus (432) Google Scholar, 4.Holmgren M. Shin K.S. Yellen G. Neuron. 1998; 21: 617-621Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar, 5.del Camino D. Yellen G. Neuron. 2001; 32: 649-656Abstract Full Text Full Text PDF PubMed Scopus (228) Google Scholar) and the G protein-activated Kir3.x (6.Sadja R. Smadja K. Alagem N. Reuveny E. Neuron. 2001; 29: 669-680Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar, 7.Jin T. Peng L. Mirshahi T. Rohacs T. Chan K.W. Sanchez R. Logothetis D.E. Mol. Cell. 2002; 10: 469-481Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar) or ATP-sensitive Kir6.x (8.Loussouarn G. Makhina E.N. Rose T. Nichols C.G. J. Biol. Chem. 2000; 275: 1137-1144Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar, 9.Phillips L.R. Enkvetchakul D. Nichols C.G. Neuron. 2003; 37: 953-962Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar) inward rectifier K+ channels suggest that an intracellular gate is formed by the bundle crossing region of the channel. These studies are consistent with spin labeling measurements made in the KcsA channel that suggest that during proton activation the second transmembrane (TM2) 1The abbreviations used are: TM2transmembrane 2ANOVAanalysis of variance. domains rotate and tilt away from the axis of the pore about a pivot point in a scissoring-type motion (1.Perozo E. Cortes D.M. Cuello L.G. Science. 1999; 285: 73-78Crossref PubMed Scopus (496) Google Scholar, 2.Liu Y.S. Sompornpisut P. Perozo E. Nat. Struct. Biol. 2001; 8: 883-887Crossref PubMed Scopus (185) Google Scholar). This work is supported by crystallization of the MthK bacterial K+ channel in the open state (10.Jiang Y. Lee A. Chen J. Cadene M. Chait B.T. MacKinnon R. Nature. 2002; 417: 523-526Crossref PubMed Scopus (1080) Google Scholar), which shows a pivot or hinge point at a highly conserved glycine residue at position 83 (equivalent to position 99 in the KcsA channel). transmembrane 2 analysis of variance. However, other parts of the channels may be associated with channel opening, because although access of large MTS reagents (such as MTSET; radius ∼2.9 Å) to the inner vestibule of a Ca2+-activated K+ channel (SK) is state-dependent, smaller MTS reagents (such as MTSEA; radius ∼1.8 Å, compared with K+ radius of 1.33 Å) can access as far as the selectivity filter equally well in open and closed channel states (11.Bruening-Wright A. Schumacher M.A. Adelman J.P. Maylie J. J. Neurosci. 2002; 22: 6499-6506Crossref PubMed Google Scholar). Similarly, access of large MTS reagents to a cyclic nucleotide-gated channel (CNG1) is gated, while Ag+ (radius 1.27 Å) has state-independent access (12.Flynn G.E. Zagotta W.N. Neuron. 2001; 30: 689-698Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar). This suggests that, in some channels at least, the bundle crossing may not be sufficient to impede the passage of permeant ions through the pore. On channel activation, other than the large movements of the TM2 domains that are described above, small movements at the inner portion of the selectivity filter of the KcsA channel have been observed in spin labeling experiments (1.Perozo E. Cortes D.M. Cuello L.G. Science. 1999; 285: 73-78Crossref PubMed Scopus (496) Google Scholar). It is therefore possible that this region of the channel may also act as a gate. Other observations are consistent with this. For example, C-type inactivation, which occurs in many voltage-gated K+ channels, closes the channel by constriction of the outer mouth of the selectivity filter (13.Lopez-Barneo J. Hoshi T. Heinemann S.H. Aldrich R.W. Recept. Channels. 1993; 1: 61-71PubMed Google Scholar, 14.Yellen G. Sodickson D. Chen T.Y. Jurman M.E. Biophys. J. 1994; 66: 1068-1075Abstract Full Text PDF PubMed Scopus (247) Google Scholar, 15.Liu Y. Jurman M.E. Yellen G. Neuron. 1996; 16: 859-867Abstract Full Text Full Text PDF PubMed Scopus (401) Google Scholar). Also, mutations within the selectivity filter of the Kir2.1 (16.Guo L. Kubo Y. Recept. Channels. 1998; 5: 273-289PubMed Google Scholar, 17.Lu T. Ting A.Y. Mainland J. Jan L.Y. Schultz P.G. Yang J. Nat. Neurosci. 2001; 4: 239-246Crossref PubMed Scopus (116) Google Scholar) and Kir6.2 (18.Proks P. Capener C.E. Jones P. Ashcroft F.M. J. Gen. Physiol. 2001; 118: 341-353Crossref PubMed Scopus (79) Google Scholar) inward rectifier K+ channels or exchanging the P-loop of the Kir2.1 channel with that of the Kir1.1b channel (19.Choe H. Palmer L.G. Sackin H. Biophys. J. 1999; 76: 1988-2003Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar) alter the kinetics of single channel flickery openings. Furthermore, a naturally occurring mutation within the selectivity filter of the G protein-activated Kir3.2 channel (Weaver mutation) alters channel sensitivity to agonist and G protein activation (20.Kofuji P. Hofer M. Millen K.J. Millonig J.H. Davidson N. Lester H.A. Hatten M.E. Neuron. 1996; 16: 941-952Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar, 21.Slesinger P.A. Patil N. Liao Y.J. Jan Y.N. Jan L.Y. Cox D.R. Neuron. 1996; 16: 321-331Abstract Full Text Full Text PDF PubMed Scopus (225) Google Scholar). In this study, we show that mutations that alter the structure of the selectivity filter affect the response of the G protein-activated Kir3.1/Kir3.4 channel to agonist. While some mutations within the selectivity filter had minimal effects, disruption of a salt bridge that maintains tension in the selectivity filter abolished agonist activation. We describe a correlation between selectivity, which is determined by the selectivity filter, and agonist activation that suggests that agonist activation of the Kir3.1/Kir3.4 channel may occur at the selectivity filter. We also address the role of a putative intracellular gate in inward rectifier K+ channels (as suggested by the KirBac1.1 crystal structure, Ref. 22.Kuo A. Gulbis J.M. Antcliff J.F. Rahman T. Lowe E.D. Zimmer J. Cuthbertson J. Ashcroft F.M. Ezaki T. Doyle D.A. Science. 2003; PubMed Google Scholar), formed by a ring of bulky hydrophobic residues. We show that these residues also contribute to agonist activation of the channel in a way that may be coupled to the gate at the selectivity filter. Molecular Biology—Mutations in the Kir3.1 or Kir3.4 channel subunits were made using site-directed PCR mutagenesis and confirmed by sequencing. Plasmids (pTLNII or pGES) containing wild-type or mutant Kir3.1 or Kir3.4 channel subunits or the hD2 (human dopamine) or M2 (muscarinic acetylcholine (ACh)) receptor (required for agonist activation of the channel) were linearized using MluI or NotI (New England Biolabs, Beverly, MA) and transcribed in vitro using SP6 or T7 RNA polymerase (Riboprobe®; Promega, Madison, WI). Electrophysiology—Xenopus oocytes were prepared as described previously (23.Lancaster M.K. Dibb K.M. Quinn C.C. Leach R. Lee J.K. Findlay J.B. Boyett M.R. J. Biol. Chem. 2000; 275: 35831-35839Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar). Oocytes were injected with 50 nl of cRNA encoding wild-type or mutant Kir3.1 (30 ng/μl) and Kir3.4 (30 ng/μl) as well as hD2 (3.8 ng/μl). In a few experiments, M2 (3.8 ng/μl) was injected instead of hD2; there was no difference in the behavior of the wild-type Kir3.1/Kir3.4 channel with the hD2 and M2 receptors (and relevant agonist). In the case of the mutations, Kir3.4[E145Q], Kir3.4[R155E], Kir3.4[E145R,R155E], Kir3.4[T148A], Kir3.1[F181A], and Kir3.4[F187A], cRNA was injected at 10 times the concentration to obtain adequate currents. Oocytes were incubated for 36–96 h at 19 °C in Barth's medium (in mm): 88 NaCl, 1 KCl, 2.4 NaHCO3, 0.82 MgSO4, 0.33 Ca(NO3)2, 0.41 CaCl2, 20 HEPES, 1.25 sodium pyruvate, 0.1 mg/ml neomycin (Sigma, Poole, UK), 100 units/0.1 mg/ml penicillin/streptomycin mix (Sigma), pH 7.4 (NaOH). Currents were recorded using the two-electrode voltage clamp technique using a GeneClamp 500B amplifier (Axon Instruments, Union City, CA) filtering at 500 Hz and sampling at 2 kHz. Voltage protocols were generated using pClamp software (Axon Instruments) with a Digidata 1200 D/A converter (Axon Instruments). Electrodes were filled with 3 m KCl (tip resistance, 1–3 MΩ). Experiments were performed at 20–25 °C. Recordings were made in solution consisting of (mm): 90 KCl, 2 CaCl2, 5 HEPES, pH 7.4 (KOH). Oocytes were placed in a small bath (∼5 × 5 × 4 mm; ∼100 μl in volume) and were directly perfused at ∼5 ml/min with a large bore manifold (∼1 mm in diameter) placed ∼1 mm from the cell. To record current-voltage relationships, currents were recorded firstly in the absence of the agonist dopamine and then following 30 s perfusion of solution containing 10 μm dopamine (along with 10 μm ascorbic acid to prevent dopamine oxidation). Oocytes were held at 0 mV and 750 ms voltage pulses were applied from -130 to + 60 mV in 10 mV increments. Currents were measured at the end of each voltage pulse. To study Rb+ permeation, the 90 mm KCl in the solution was replaced with 90 mm RbCl (pH titrated using RbOH). To measure the rate of channel activation on application of agonist and the effect of Gαi3, solution without agonist was rapidly exchanged for solution containing 10 μm dopamine (to measure channel activation) or 10 μm ACh (to measure the effect of Gαi3) and current was recorded continuously at -80 mV. To test the ability of our perfusion system to rapidly change the solution around each oocyte, we measured the rate of current activation in the constitutively active Kir3.1[S170P]/Kir3.4[S176P] mutant channel on switching from ND96 to solution containing 90 mm K+. Current activated in 0.7 ± 0.1 s (n = 5) and this is >10x faster than current activation on application of dopamine. In all experiments with mutant channels, data were also collected from the wild-type channel expressed in the same batch of oocytes. Data analysis was performed using Clampfit (Axon Instruments) and SigmaPlot (SPSS Science, Chicago, IL) software. Data are given as means ± S.E. (n = number of oocytes). Statistical analysis was performed using a Student's t test or ANOVA as appropriate. p values of <0.05 were considered to signify a significant difference. Modeling—A comparative model of the Kir3.1/Kir3.4 tetrameric channel was constructed based on the crystal structure of KcsA as described previously (24.Claydon T.W. Rose T. Nichols C.G. Boyett M.R. Biophys. J. 2003; 84: 80aGoogle Scholar, 25.Dibb K.M. Makary S.Y. Leach R. Rose T. Nichols C.G. Boyett M.R. Biophys. J. 2003; 84: 80aGoogle Scholar). Effect of Mutation of the Bulky Hydrophobic Residues at the Bundle Crossing—The comparative model of the Kir3.1/Kir3.4 channel (Fig. 1) shows a narrowing of the permeation pathway at the bundle crossing formed by the large side chains of the phenylalanine residues at position 181 in Kir3.1 and 187 in Kir3.4 (Fig. 1, A and C). The recently crystallized KirBac1.1 bacterial inward rectifier K+ channel also possesses phenylalanine residues at the equivalent position (F146 (22.Kuo A. Gulbis J.M. Antcliff J.F. Rahman T. Lowe E.D. Zimmer J. Cuthbertson J. Ashcroft F.M. Ezaki T. Doyle D.A. Science. 2003; PubMed Google Scholar)). Because their large hydrophobic side chains are ideally positioned to prevent movement of water through the narrow bundle crossing, the four phenylalanine residues in KirBac1.1 were proposed to be involved in channel activation (22.Kuo A. Gulbis J.M. Antcliff J.F. Rahman T. Lowe E.D. Zimmer J. Cuthbertson J. Ashcroft F.M. Ezaki T. Doyle D.A. Science. 2003; PubMed Google Scholar). We have investigated the role of these phenylalanine residues in agonist activation of the Kir3.1/Kir3.4 channel. Fig. 2 shows the effect of replacement of the phenylalanine residues with either methionine residues, the equivalent residue in the constitutively active Kir2.1 channel, or alanine residues, which have relatively small hydrophobic side chains. The left hand panel shows currents through the wild-type channel recorded in the presence of 90 mm K+ during 750 ms voltage pulses to -130 to +60 mV from a holding potential of 0 mV. The top set of traces was recorded in the absence of agonist, the middle set was recorded in the presence of agonist (10 μm dopamine) and the bottom set shows the agonist-activated current, the difference in current with and without agonist. Mean current-voltage relationships are also shown. In the absence of agonist, current was recorded through the wild-type channel (Fig. 2). Basal current is thought to be due to a high level of free Gβγ and/or a low level of Gαi within the Xenopus oocyte (26.Peleg S. Varon D. Ivanina T. Dessauer C.W. Dascal N. Neuron. 2002; 33: 87-99Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar). Nevertheless, wild-type current was substantially increased, by 60.7 ± 6.4%, on application of agonist (Fig. 2). Replacement of both pairs of phenylalanine residues with methionine residues (Kir3.1[F181M]/Kir3.4[F187M]) had no effect on the agonist dependence of the channel. This can be seen in Fig. 2, which shows current traces and mean current-voltage relationships for the mutant channel, and also in Fig. 3A (bar 4), which shows the agonist-activated current (at -130 mV) of various mutant channels normalized to that of the wild-type channel recorded from the same batch of oocytes. Although mutation to a methionine residue in all four subunits had no effect, mutation in either Kir3.1 or Kir3.4 alone did have a modest effect on agonist dependence (Fig. 3A, bars 2 and 3). The mutation, Kir3.1[F181M], reduced agonist activation by 30.9 ± 2.3% (n = 15; ANOVA, p < 0.05) compared with that of the wild-type channel, while the mutation, Kir3.4[F187M], increased agonist activation by 42.1 ± 3.9% (n = 5; ANOVA, p < 0.05) compared with that of the wild-type channel (Fig. 3A, bars 2 and 3). The effect of replacement of the phenylalanine residues with small alanine residues (Kir3.1[F181A]/Kir3.4[F187A]) was much greater (Figs. 2 and 3A, bar 5). The double mutation, Kir3.1[F181A]/Kir3.4[F187A], abolished agonist activation; the agonist-activated current was reduced by 114 ± 4% (n = 6; ANOVA, p < 0.05) compared with that of the wild-type channel. In proline-scanning experiments of the TM2 domain (7.Jin T. Peng L. Mirshahi T. Rohacs T. Chan K.W. Sanchez R. Logothetis D.E. Mol. Cell. 2002; 10: 469-481Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar), introduction of proline residues affected agonist activation with an α-helical periodicity (residues highlighted in blue in Fig. 1D). The double mutation, Kir3.1[S170P]/Kir3.4[S176P], had the greatest effect on agonist activation and produced this by introducing a kink into the helix of the TM2 domains (7.Jin T. Peng L. Mirshahi T. Rohacs T. Chan K.W. Sanchez R. Logothetis D.E. Mol. Cell. 2002; 10: 469-481Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar). In the present study, the double mutation, Kir3.1[S170P]/Kir3.4[S176P], reduced agonist activation by 80.5 ± 4.9% (Fig. 2; n = 5, ANOVA, p < 0.05) compared with that of the wild-type channel (Fig 3A, bar 6). As a control, we investigated the effect of the mutation of an aspartate residue within the TM2 domain (D173 in Kir3.1; equivalent to D172 in Kir2.1) that is thought to be involved with polyamine block of inward rectifier K+ channels. The mutation, Kir3.1[D173Q], had no effect on agonist activation; agonist-activated current through the Kir3.1[D173Q]/Kir3.4 mutant channel was 93.1 ± 1.0% that of the wild-type channel (n = 2; ANOVA, not significant). Mutations that Disrupt the Selectivity Filter Abolish Agonist Activation—Fig. 1B shows an enlarged view of the part of the P-loop that forms the selectivity filter of the Kir3.1/Kir3.4 channel. As in the Kir2.1 channel (27.Yang J. Yu M. Jan Y.N. Jan L.Y. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 1568-1572Crossref PubMed Scopus (100) Google Scholar), a salt bridge is thought to exist behind the selectivity filter between the negatively charged glutamate residue at position 139 (Glu-139) and the positively charged arginine residue at position 149 (Arg-149) in the Kir3.1 subunit and between Glu-145 and Arg-155 in the Kir3.4 subunit (see also the alignment in Fig. 1D). Fig. 4 shows the effect of agonist application on mutant Kir3.1/Kir3.4 channels in which the salt bridge behind the selectivity filter had been broken by neutralizing or reversing the charges at Glu-139/Glu-145 (in Kir3.1 and Kir3.4, respectively) and/or Arg-155 (in Kir3.4). Currents were recorded under the same conditions as described in Fig. 2. Disruption of the salt bridge in the Kir3.4 subunit (but not the Kir3.1 subunit, see below), by the Kir3.4[E145Q], Kir3.4[R155E] and Kir3.4[E145R,R155E] mutations, had dramatic effects on agonist activation. Current through these mutant channels was not activated by agonist, and the channels were constitutively active (Fig. 4). Again, this can also be seen in Fig. 3A (bars 8–10). The agonist-activated current was reduced by 89.5 ± 1.9, 109.4 ± 11.2 and 120.4 ± 1.3% in the Kir3.1/Kir3.4[E145Q], Kir3.1/Kir3.4[R155E] and Kir3.1/Kir3.4[E145R,R155E] mutant channels, respectively (n = 5–6; ANOVA, p < 0.05) when compared with that of the wild-type channel. Interestingly, only breaking the salt bridge in the Kir3.4 subunit made the channel constitutively active. Breaking the salt bridge in Kir3.1, by the mutation Kir3.1[E139Q], did not affect agonist activation (Fig. 3A, bar 7). To investigate the effect of disrupting the salt bridge on channel activation further, we measured the sensitivity of the wild-type channel and the Kir3.1/Kir3.4[E145Q] mutant channel (taken as an example of a mutant channel that lacks agonist activation) to Gβγ activation. Fig. 5A shows current through the wild-type channel and the Kir3.1/Kir3.4[E145Q] mutant channel recorded with and without injection of 3 ng/oocyte of Gβγ. Injection of 3 ng/oocyte of Gβγ activated the wild-type channel, but had no effect on the Kir3.1/Kir3.4[E145Q] mutant channel. Panels B and C of Fig. 5 show mean current-voltage relationships and dose-response curves for a range of Gβγ concentrations. At each concentration tested, Gβγ activated the wild-type channel, but failed to activate the Kir3.1/Kir3.4[E145Q] mutant channel (Fig. 5, B and C). These data show that the Kir3.1/Kir3.4[E145Q] mutant channel, in which the salt bridge has been broken, is insensitive to Gβγ activation as well as to agonist activation. Thus far, we have described mutant channels that do not respond to agonist, such as Kir3.1/Kir3.4[E145Q], as being constitutively active. However, it is possible that these channels are instead highly sensitive to Gβγ. Mutant channels may be maximally activated by endogenous Gβγ and so do not respond to agonist. To distinguish between these two possibilities, we co-expressed Gαi3, which sequesters endogenous Gβγ and abolishes basal current through the wild-type channel (26.Peleg S. Varon D. Ivanina T. Dessauer C.W. Dascal N. Neuron. 2002; 33: 87-99Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar). Fig. 6A shows continuous current recordings at -80 mV from oocytes injected with wild-type or Kir3.1/Kir3.4[E145Q] mutant channels in the absence or presence of 3 ng/oocyte of Gαi3. These experiments were performed with the M2 muscarinic ACh receptor. In the absence of Gαi3, substantial basal current was recorded through the wild-type channel and on application of agonist (10 μm ACh), current was increased (Fig. 6A, left as observed previously e.g.Fig. 2). Co-expression of Gαi3 abolished basal current, but not the agonist-activated current, through the wild-type channel (Fig. 6A, left). In contrast, basal current through the Kir3.1/Kir3.4[E145Q] mutant channel was not affected by Gαi3 (Fig. 6A, right). Furthermore, in the absence and presence of Gαi3, agonist application still failed to activate the channel (Fig. 6A, right). These results are confirmed by the mean data in Fig. 6B, which shows mean current amplitudes in the absence and presence of ACh for the wild-type and mutant channels with and without Gαi3. These data suggest that the Kir3.1/Kir3.4[E145Q] mutant channel is indeed constitutively active and not highly sensitive to Gβγ. Also highlighted in Fig. 1B are the alanine residue at position 142 in Kir3.1 and the threonine residue that is in the equivalent position in Kir3.4 (Thr-148). In Kir2.1, this site has been shown to be important in high affinity block of the channel by Cs+, Rb+, and Ba2+ (28.Thompson G.A. Leyland M.L. Ashmole I. Sutcliffe M.J. Stanfield P.R. J. Physiol. 2000; 526: 231-240Crossref PubMed Scopus (58) Google Scholar, 29.Alagem N. Dvir M. Reuveny E. J. Physiol. 2001; 534: 381-393Crossref PubMed Scopus (71) Google Scholar). Fig. 7 shows the effect of mutation at this site on agonist activation. Replacement of the alanine residue in Kir3.1 with a threonine residue (Kir3.1[A142T]; Fig. 7left), or the threonine residue in Kir3.4 with an alanine residue (Kir3.4[T148A]; Fig. 7middle) significantly reduced the extent of channel activation on application of agonist. This effect was modest in the case of the Kir3.1[A142T]/Kir3.4 mutant channel and large in the case of the Kir3.1/Kir3.4[T148A] mutant channel. Fig. 3A (bars 11 and 12) shows that the agonist-activated current at -130 mV was reduced by 32.6 ± 3.8% and 61.5 ± 1.3%, in the case of the Kir3.1[A142T]/Kir3.4 and Kir3.1/Kir3.4[T148A] mutant channels, respectively, (n = 5; ANOVA, p < 0.05) when compared with that of the wild-type channel. Curiously, the double mutation, Kir3.1[A142T]/Kir3.4[T148A], had no effect on agonist activation (Fig. 3A, bar 13; Fig. 7; n = 5; not significant). Correlation between Agonist Activation and Selectivity—The salt bridge behind the selectivity filter is likely to be important for the correct conformation of the selectivity filter (30.Berneche S. Roux B. Biophys. J. 2000; 78: 2900-2917Abstract Full Text Full Text PDF PubMed Scopus (290) Google Scholar). The dramatic effect of disruption of the salt bridge in Kir3.4 could, therefore, be the result of the disruption of the selectivity filter, rather than the salt bridge per se. It is possible that the effects of the mutation of Ala-142 and Thr-148 in Kir3.1 and Kir3.4 may again be the result of disruption of the selectivity filter. To test this, the effect of the various mutations on channel selectivity as well as agonist activation was investigated. Fig. 3B shows a measure of channel selectivity. In these experiments, the 90 mm K+ in the extracellular solution was replaced with 90 mm Rb+. Rb+ permeated the wild-type channel to a certain extent; at -130 mV, Rb+ current was 0.35 ± 0.01 (n = 5) the size of the K+ current. However, in the case of the mutant channels in which the salt bridge in Kir3.4 had been broken and in the case of the Kir3.1/Kir3.4[T148A] mutant channel, Rb+ permeation was dramatically increased; Rb+ current was 0.88 ± 0.06, 1.33 ± 0.07, 0.92 ± 0.02, and 0.58 ± 0.03 the size of the K+ current in Kir3.1/Kir3.4[E145Q], Kir3.1/Kir3.4[R155E], Kir3.1/Kir3.4[E145R, R155E], and Kir3.1/Kir3.4[T148A] mutant channels, respectively (n = 5–6; ANOVA, p < 0.05), suggesting that, in these channels, selectivity was reduced or abolished (see also Ref. 31.Dibb K.M. Leach R. Findlay J.B. Boyett M.R. Biophys. J. 2001; 80: 624aGoogle Scholar). Interestingly, selectivity was also altered by the double mutation, Kir3.1[F181A]/Kir3.4[F187A]; Rb+ current was 0.64 ± 0.02 the size of the K+ current (n = 5; ANOVA, p < 0.05). However, selectivity was not affected in the Kir3.1[S170P]/Kir3.4[S176P] or Kir3.1[D173Q] mutant channels; Rb+ currents were 0.29 ± 0.02 and 0.32 ± 0.01 the size of the K+ current, respectively (n = 2–5; ANOVA, not significant). Fig. 3C shows that agonist dependence and channel selectivity are significantly correlated (r2 = 0.47, p < 0.02). These data suggest that the selectivity filter of the Kir3.1/Kir3.4 channel may be the agonist-activated gate. Rate of Agonist Activation—In Fig. 3C, the Kir3.1[E139Q]/Kir3.4 mutant channel (point 7) is an outlier: whereas disrupting the salt bridge in the Kir3.1 subunit by the mutation, Kir3.1[E139Q], had no effect on the size of the agonist-activated current, it did reduce selectivity; Rb+ current was 0.81 ± 0.06 the size of the K+ current (n = 5; ANOVA, p < 0.05). However, although the size of the agonist-activated current was not altered, the rate of agonist activation of the Kir3.1[E139Q]/Kir3.4 mutant channel was. Fig. 8 shows the rate of agonist activation in the wild-type channel and those mutant channels (including the Kir3.1[E139Q]/Kir3.4 mutant channel) in which agonist dependence was not completely abolished. Typical continuous current traces recorded at -80 mV during the rapid application of the agonist, dopamine, are shown in Fig. 8A. Cells were perfused initially with solution containing no agonist. This was then rapidly exchanged with solution containing dopamine. Fig. 8B shows the mean 10–90% rise time of agonist-activated current in the wild-type and mutant channels. Wild-type current activated in 7.7 ± 0.6 s (n = 13) on application of agonist. Activation was significantly slowed by the mutation Kir3.1[E139Q]; current activated in 11.1 ± 1.2 s (n = 6; ANOVA, p < 0.05). The rate of agonist activation was also slowed by a number of other mutations. Channels in which one of the pairs of phenylalanine residues was replaced (the Kir3.1[F181M]/Kir3.4 and Kir3.1/Kir3.4[F187M] mutant channels) activated more slowly on application of agonist; current activated in 10.8 ± 1.1 s and 12.0 ± 1.0 s, respectively (n = 4–5; ANOVA, p < 0.05). Also the mutations Kir3.4[T148A] and Kir3.1[A142T]/Kir3.4[T148A] slowed channel activation; current activated within 13.6 ± 0.6 s and 12.0 ± 0.3 s, respectively (n = 5; ANOVA, p < 0.05). In contrast, mutation of both of the pairs of phenylalanine residues to methionine residues (Kir3.1[F181M]/Kir3.4[F187M]) or of the alanine residue in Kir3.1 to a threonine residue (Kir3.1[A142T]) had no effect on the rate of agonist activation (n = 6–8; not significant). Spermine Can Permeate the Channel in the Absence of Agonist—We have previously shown that polyamines, such as spermine, can permeate the Kir3.1/Kir3.4 channel (32.Makary S.Y. Dibb K.M. Claydon T.W. Leach R. Nichols C.G. Boyett, Pflügers Arch. 2002; 443: S170Google Scholar, 33.Claydon T.W. Makary S.Y. Boyett M.R. J. Physiol. 2002; 544: 11PGoogle Scholar, 34.Makary S.Y. Claydon T.W. Boyett M.R. Biophys. J. 2003; 84: 314aAbstract Full Text Full Text PDF PubMed Scopus (7) Google Scholar). F
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