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Inhibition of human ether a go-go potassium channels by Ca2+/calmodulin

2000; Springer Nature; Volume: 19; Issue: 13 Linguagem: Inglês

10.1093/emboj/19.13.3263

1460-2075

Roland Schönherr,

Cardiac electrophysiology and arrhythmias

Article3 July 2000free access Inhibition of human ether à go-go potassium channels by Ca2+/calmodulin Roland Schönherr Roland Schönherr Arbeitsgruppe Molekulare und zelluläre Biophysik, am Klinikum der Friedrich-Schiller-Universität Jena, Drackendorfer Straße 1, D-07747 Jena, Germany Search for more papers by this author Karsten Löber Karsten Löber Arbeitsgruppe Molekulare und zelluläre Biophysik, am Klinikum der Friedrich-Schiller-Universität Jena, Drackendorfer Straße 1, D-07747 Jena, Germany Search for more papers by this author Stefan H. Heinemann Corresponding Author Stefan H. Heinemann Arbeitsgruppe Molekulare und zelluläre Biophysik, am Klinikum der Friedrich-Schiller-Universität Jena, Drackendorfer Straße 1, D-07747 Jena, Germany Search for more papers by this author Roland Schönherr Roland Schönherr Arbeitsgruppe Molekulare und zelluläre Biophysik, am Klinikum der Friedrich-Schiller-Universität Jena, Drackendorfer Straße 1, D-07747 Jena, Germany Search for more papers by this author Karsten Löber Karsten Löber Arbeitsgruppe Molekulare und zelluläre Biophysik, am Klinikum der Friedrich-Schiller-Universität Jena, Drackendorfer Straße 1, D-07747 Jena, Germany Search for more papers by this author Stefan H. Heinemann Corresponding Author Stefan H. Heinemann Arbeitsgruppe Molekulare und zelluläre Biophysik, am Klinikum der Friedrich-Schiller-Universität Jena, Drackendorfer Straße 1, D-07747 Jena, Germany Search for more papers by this author Author Information Roland Schönherr1, Karsten Löber1 and Stefan H. Heinemann 1 1Arbeitsgruppe Molekulare und zelluläre Biophysik, am Klinikum der Friedrich-Schiller-Universität Jena, Drackendorfer Straße 1, D-07747 Jena, Germany *Corresponding author. E-mail: [email protected] The EMBO Journal (2000)19:3263-3271https://doi.org/10.1093/emboj/19.13.3263 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Intracellular Ca2+ inhibits voltage-gated potassium channels of the ether à go-go (EAG) family. To identify the underlying molecular mechanism, we expressed the human version hEAG1 in Xenopus oocytes. The channels lost Ca2+ sensitivity when measured in cell-free membrane patches. However, Ca2+ sensitivity could be restored by application of recombinant calmodulin (CaM). In the presence of CaM, half inhibition of hEAG1 channels was obtained in 100 nM Ca2+. Overlay assays using labelled CaM and glutathione S-transferase (GST) fusion fragments of hEAG1 demonstrated direct binding of CaM to a C-terminal domain (hEAG1 amino acids 673–770). Point mutations within this section revealed a novel CaM-binding domain putatively forming an amphipathic helix with both sides being important for binding. The binding of CaM to hEAG1 is, in contrast to Ca2+-activated potassium channels, Ca2+ dependent, with an apparent KD of 480 nM. Co-expression experiments of wild-type and mutant channels revealed that the binding of one CaM molecule per channel complex is sufficient for channel inhibition. Introduction The role of potassium channels (K+ channels) in the generation of action potentials and the electrical signalling of excitable cells is well documented (Hille, 1992). They contribute to the setting of the resting membrane potential, a function that is not restricted to excitable cells. Many K+ channel types are subject to various regulation processes that couple them to intracellular signalling pathways. One prominent example is the opening of calcium-activated K+ channels (KCa) by cytosolic Ca2+. At least for two classes of KCa channels (SK and IK), it has been suggested that calmodulin (CaM) forms a constitutive part of these channels (Xia et al., 1998; Fanger et al., 1999). The resulting CaM-gated channels open in situations where the Ca2+ concentration is strongly elevated above basal levels. In contrast to KCa channels, the K+ channels encoded by the ether à go-go (EAG) gene are activated by membrane depolarization and inhibited by intracellular Ca2+ (Stansfeld et al., 1996), with a half-maximal inhibition concentration of ∼100 nM. This means that EAG channels are ready to be activated at typical [Ca2+]i but undergo Ca2+-mediated inhibition in situations where KCa channels start to become activated. Although expression patterns of EAG have been evaluated in different species, the functional role of this channel is only beginning to be understood. The strong expression in rat brain (Ludwig et al., 1994) can be considered an indication for a prominent role in neuronal signalling. Likewise, expression of a recently described isoform (rEAG2; Saganich et al., 1999) was restricted mainly to the brain, but with a pattern distinct from that of rEAG1. Despite the lack of specific pharmacological tools for channels of the EAG family, EAG-mediated currents can be identified reliably based on unique functional properties. Voltage-dependent activation of EAG channels is strongly dependent on the holding potential and is regulated by extracellular divalent cations (Ludwig et al., 1994; Terlau et al., 1996). This feature led to the identification of EAG channels in human myocytes, where they play a role in the formation of muscle fibres (Occhiodoro et al., 1998). Apparently, EAG expression in myoblasts is a prerequisite for these cells to lower their resting potential and thereby to gain fusion competence (Bijlenga et al., 1998). Another role has become apparent after the identification of EAG channels in various cancer cell lines (Meyer and Heinemann, 1998; Meyer et al., 1999; Pardo et al., 1999). In human neuroblastoma cells, hEAG1 expression is dependent on the cell cycle (Meyer and Heinemann, 1998), and rat EAG channels, expressed in Xenopus oocytes, showed cell cycle-related functional changes triggered by the mitosis-promoting factor (Brüggemann et al., 1997; Pardo et al., 1998). These results suggest a role for hEAG in tumour progression, strongly supported by the recent work of Pardo et al. (1999), showing enhanced cell growth of hEAG-transfected cells in a mouse model for tumour progression. A somewhat similar function may be assigned to KCa channels, as inhibition of IK channels in human T lymphocytes leads to inhibition of the proliferative T-cell response after activation (Jensen et al., 1999). Mechanisms by which both KCa channels and EAG channels influence the cell cycle remain to be elucidated, but the regulation of both channel types by intracellular Ca2+ suggests a functional link. Here we describe the molecular basis for the inhibition of the delayed rectifier channel hEAG1 by Ca2+/CaM. Results hEAG1 channels expressed in Xenopus oocytes hEAG1 channels were expressed in oocytes of Xenopus laevis. For an assessment of the effect of intracellular Ca2+ on the channels, it was desirable to obtain stable inside-out patch configurations. In solutions containing high concentrations of aspartate instead of chloride (see Materials and methods), we could record from cell-free patches for >30 min without significant rundown. The analysis of the voltage dependence of channel activation (Figure 1A and B) as well as the pre-pulse dependence of channel activation kinetics (Figure 1C) revealed that hEAG1 channels, measured in excised patches of Xenopus oocytes, show the same properties as bovine, rat and human EAG channels in mammalian cells (Ludwig et al., 1994; Frings et al., 1998; Occhiodoro et al., 1998). Upon patch excision (Figure 1B), an increase in current amplitude of variable magnitude was often observed, indicating the removal of channel block by intracellular Na+ (Pardo et al., 1998) and/or Ca2+. Figure 1.Recording of hEAG1-mediated currents from cell-free membrane patches. (A) Current responses to depolarizing pulses (protocol on top) were recorded from an inside-out patch of Xenopus oocytes injected with mRNA coding for hEAG1. (B) The maximum current amplitudes after 500 ms were plotted as a function of the test voltage (open circles). Also shown is the result of current recordings in the on-cell mode of the same patch (filled circles), indicating a small increase in current upon membrane excision. Straight lines connect the data points. (C) Current recordings for assaying the dependence of activation kinetics on the holding voltage (see protocol). The depolarizations to +50 mV activate hEAG1 channels with quite different time courses depending on the holding voltage applied for 5 s. Download figure Download PowerPoint Ca2+ sensitivity requires calmodulin The sensitivity of hEAG1 channels to intracellular Ca2+ is easily observed when a membrane patch is excised into bath solutions containing free Ca2+. In Figure 2A, the response of hEAG1 channels to a change from cytosolic [Ca2+] of an intact oocyte (on-cell patch) to 200 nM free Ca2+ (inside-out patch) is shown. Excision of the patch resulted in an almost complete reduction of the current response to depolarizing pulses. The current was restored completely by subsequent perfusion of the patch with an EGTA-buffered Ca2+-free solution. Upon a second application of 200 nM Ca2+ to the same patch, however, no current reduction was observed, indicating the loss of a cytosolic factor during the perfusion in the absence of Ca2+. Brief cramming of the patch pipette back into the oocyte to expose the patch membrane to the cytosol immediately restored the Ca2+ sensitivity of the channel and abolished the current. To investigate the nature of the required cytosolic factor, we repeated the experiment in the inside-out configuration and applied cell lysate. The first trace in Figure 2B was recorded in 1 μM Ca2+ after perfusion of the excised patch with Ca2+-free solution. Adding the lysate of IGR1 human melanoma cells, which natively express hEAG1 channels (Meyer et al., 1999), in the vicinity of the patch pipette resulted in a strong current reduction. Lysate from Xenopus oocytes had the same effect (data not shown). The lysate could be inactivated by pre-incubation with a synthetic peptide corresponding to the CaM-binding domain of CaM kinase II (Figure 2B). The channels only became sensitive again to the untreated lysate plus Ca2+ after thorough washing with Ca2+-free solution, indicating that no irreversible channel modification resulted from the application of the CaM-binding peptide. From this experiment, we concluded that CaM must be at least one obligate component of the cell lysate required to confer Ca2+ sensitivity to the channels. The experiment shown in Figure 2C illustrates that purified His-tagged CaM is sufficient to cause Ca2+-induced inhibition of hEAG1 channels. A membrane patch was excised directly into a solution without free Ca2+. The small current increase in the inside-out patch (second trace) compared with the on-cell situation (first trace) reflects the partial inhibition of the channels by cytosolic Ca2+ in the intact oocyte. Upon perfusion of this patch with CaM in the presence of 200 nM Ca2+, hEAG current was almost completely blocked. The block was relieved by perfusion with solution containing CaM in the absence of Ca2+, confirming that both CaM and Ca2+ are required. Figure 2.Calmodulin is required for current inhibition. hEAG1-mediated currents were recorded from on-cell or inside-out patches of oocyte membranes in 400 ms depolarizations to +40 mV with a repetition interval of 10 s. Each trace represents the last of five pulses under identical conditions. The cartoons indicate the patch configuration; free Ca2+ concentrations are given below the traces. (A) After stabilization of the current amplitude in the on-cell configuration (first trace), the patch was excised into the bath solution, containing 200 nM free Ca2+. Apparently the current was reduced substantially by 200 nM Ca2+, but recovered to the original amplitude after wash with Ca2+-free solution. However, it could not be blocked again by subsequent application of 200 nM Ca2+. Pushing back the pipette tip with the membrane patch into the cytosol of the oocyte restored the Ca2+ sensitivity of the channels (n >15). (B) The Ca2+ sensitivity could also be restored by application of cell lysate (here lysate from human melanoma cells, IGR1) to the patch. In this panel, all shown traces were obtained from an inside-out patch excised into a bath solution containing 1 μM free Ca2+. The patch had been washed previously without Ca2+ to remove the Ca2+ sensitivity of the channels. When cell lysate (10 μl) was pipetted close to the patch pipette, channels were inactivated (second trace). Application of the same lysate after pre-incubation with a CaM-binding peptide (CaM kinase II, residues 290–309) at a concentration of 100 μM restored the current, suggesting that CaM is a required factor in the lysate. Re-administration of untreated lysate after a wash without lysate rendered the channels Ca2+ sensitive again (last trace) (n = 2). (C) Purified recombinant CaM was applied to an excised Ca2+-insensitive patch, resulting in immediate current block by 200 nM Ca2+. The effect of CaM on the channels was Ca2+ dependent, as it did not inactivate channels in the absence of Ca2+ (fourth trace) (n = 20). Download figure Download PowerPoint Localization of a CaM-binding domain To address the question of whether CaM binds directly to the hEAG1 channel, gene fusions were constructed, linking parts of the heag1 gene to the glutathione S-transferase (GST) gene. In overlay assays, [35S]methionine-labelled human CaM ([35S]CaM) was incubated with various GST fusion proteins immobilized on a polyvinylidene (PVDF) membrane. For this purpose, the fusion proteins were expressed in Escherichia coli and loaded directly onto an SDS–polyacrylamide gel without purification (Figure 3C). A construct covering the complete C-terminus of hEAG1 (GST–12345, Figure 3B) showed weak, but reproducible binding of [35S]CaM (Figure 3D, first lane). To narrow down the binding site within the C-terminus, a series of truncated GST fusions was constructed as depicted in Figure 3B. A fragment containing 97 amino acids from the middle of the C-terminus (GST–3), as well as all larger fragments containing this part gave positive signals in the overlay assay (Figure 3C and D). Constructs lacking this part of the C-terminal domain (GST–12 and GST–45) did not interact with CaM. In this experiment, as well as in several independent experiments, the positive fragments resulted in signals of variable intensity. This may reflect variability in the exposure of the binding site within the immobilized polypeptides, which is certainly influenced by the neighbouring domains. The generally weaker signals obtained with the largest fusions (GST–1234 and GST–12345) may, in addition, be caused by a weaker expression and by incomplete transfer to the blotting membrane. Figure 3.Localization of the CaM-binding site in the C-terminus of hEAG1. (A) Transmembrane topology of an α-subunit of hEAG1. (B) Sectioning of the C-terminal domain of hEAG1 and the corresponding names of the GST fusion proteins. Also indicated are the residue numbers of the sites of deletion as well as an indicator of CaM binding (±, see below). (C) A Coomassie-stained gel of lysates from E.coli cells expressing the indicated GST fusion proteins. (D) Autoradiography of a membrane overlay with radioactively labelled CaM. The same samples as shown in (C) were blotted to a PVDF membrane and incubated with [35S]CaM. Clear CaM-binding signals for the constructs GST–3, −34 and −345 and faint signals for the longer constructs GST–12345 (at ∼81 kDa) and GST–1234 (at ∼70 kDa) are visible. No signals were detected for the constructs GST–12 and GST–45. Download figure Download PowerPoint Critical residues of the CaM-binding motif To characterize the CaM-binding motif within the GST–3 fragment, several point mutations were introduced into this part of the gene. We concentrated on charged residues, replacing them by neutral amino acids, and tested the Ca2+ sensitivity of such mutants in excised patches. Out of seven mutations, five (C1–4 and C8, see Materials and methods) did not lead to remarkable changes in the Ca2+ sensitivity of the channels (data not shown). The mutations C5 and C6 resulted in channels that could not be fully blocked by 200 nM Ca2+ in the presence of CaM (Figure 4A and C). The protein sequence in the region of these two mutants fits to a common feature of many thus far determined CaM-binding motifs, which are characterized by amphiphilic helices divided into a basic and a hydrophobic side (James et al., 1995). Figure 4B shows a stretch of 13 residues, which covers the residues replaced in the C5 and C6 mutants, in a helical wheel projection. To test whether hydrophobicity in this segment is really required for CaM interaction with hEAG1, we tested a mutant in which the phenylalanine residues in positions 714 and 717 were substituted by serines. Indeed, the resulting channels (C7) showed even lower Ca2+ sensitivity than C5 and C6, as almost 100% of the initial current in 0 Ca2+ was retained after perfusion with 200 nM Ca2+ and purified CaM (Figure 4C). The same strong effect was obtained by combination of the replacements used in the mutants C5 and C6. Like C7, this channel mutant (C9) did not respond to perfusion of the patch with Ca2+/CaM. The electrophysiological results obtained with the mutant channels suggest that either binding of CaM to the channel or the inactivation mechanism triggered by Ca2+/CaM must be impaired in these mutants. Therefore, these mutations were transferred into the GST–34 construct that exhibited strong binding of labelled CaM in overlay assays (Figure 3D). Figure 4D and E shows that mutations C5 and C6 strongly reduced the binding of CaM to these GST fusions, whereas the mutants C7 and C9 did not bind CaM. These data suggest that the residues altered in the mutants C5, C6, C7 and C9 are part of a CaM-binding domain in hEAG1 channels that presumably forms a typical amphiphilic CaM-binding helix. The alignment shown in Figure 4A reveals noticeable analogies of this site to corresponding fragments of Drosophila EAG and the EAG2 isoform from rat (Saganich et al., 1999), as well as to postulated CaM-binding sites in hIK and rSK2 (Fanger et al., 1999; Keen et al., 1999) and in the cyclic nucleotide-gated (CNG) channel rOLFα (Liu et al., 1994). Figure 4.Characterization of a CaM-binding domain. (A) Amino acid sequence of hEAG1 between residues 707 and 726 (upper row). Indicated on top of this sequence are the residues that were substituted and the corresponding names of the resulting double site mutants. The mutant C9 is a combination of C5 + C6. The following rows show the alignment of fragments corresponding to Drosophila EAG (DDBJ/EMBL/GenBank accession No. M61157), rat EAG2 (DDBJ/EMBL/GenBank accession No. AF185637), as well as postulated CaM-binding sites of hIK and rSK2 (DDBJ/EMBL/GenBank accession Nos AF022150 and U69882) and from the cyclic nucleotide-gated channel rOLFα (DDBJ/EMBL/GenBank accession No. X55519). The first amino acid residues in the fragments shown correspond to the following positions in the full-length proteins: 707, 703, 724, 338, 450 and 61 (from top to bottom). (B) The residues of hEAG1 marked by the line in (A) (numbered from H1 to Q13) are shown as a hypothetical α-helical wheel (3.6 residues per turn); the mutated residues are shown in bold. The dashed line indicates that such a helix is amphipathic, with one side being hydrophilic, the other hydrophobic. (C) hEAG1 wild-type (wt) and the indicated mutants were expressed in Xenopus oocytes and the sensitivity of the resulting currents to Ca2+/CaM was assayed by measuring in inside-out patches the reduction of current when the bath solution was switched from Ca2+-free solution to a solution containing recombinant human CaM and 200 nM free Ca2+. Wild-type currents were reduced almost completely, whereas currents mediated by the mutants C7 and C9 were almost insensitive. Mutants C5 and C6 display an intermediate Ca2+/CaM sensitivity (n = 6 for all data points). The mutants indicated were also generated in the background of the fusion protein GST–34 (see Figure 3). (D and E) The corresponding Coomassie-stained protein gel and the CaM overlay blots, respectively. Download figure Download PowerPoint Ca2+ dependence of channel block and CaM binding In human melanoma cells expressing hEAG1, we previously determined an IC50 value of 99 ± 29 nM for channel inhibition by Ca2+i (Meyer et al., 1999). To test whether or not the excised patch assay reflects the native situation with respect to the Ca2+ dependence, varying Ca2+ and fixed CaM concentrations were applied to inside-out membrane patches containing hEAG1 channels. The first trace in Figure 5A shows the current response in the absence of Ca2+ and CaM. The following traces were recorded with varying [Ca2+] and constant [CaM]. The results from 12 patches are summarized in Figure 5C, where we plotted normalized current amplitudes over [Ca2+] (open circles). Data fits to a Hill function yielded an apparent IC50 value of 106 ± 4 nM and a Hill coefficient of 4.2 ± 0.2. To address the question of whether this Ca2+ regulation is determined by the binding properties of CaM, we performed in vitro binding assays using the same solutions as in the patch experiments. CaM was incubated with purified GST–34 fusion protein and the amount of CaM that could be co-precipitated on the binding matrix for GST was estimated from SDS–polyacrylamide gels (Figure 5B). The relative intensities of the CaM protein bands in the gels were plotted versus [Ca2+] in Figure 5C (filled symbols); a Hill function fitted to these data points yielded an apparent KD value of 480 ± 40 nM Ca2+ and a Hill coefficient of 1.7 ± 0.2. Figure 5.Concentration dependence of current block and CaM binding. (A) The dependence of hEAG1 current inhibition by Ca2+/CaM on the concentration of free Ca2+ was measured in inside-out patches in various solutions containing identical concentrations of CaM, but different concentrations of Ca2+. The current amplitudes were normalized to the control value in Ca2+-free solution and were plotted as a function of [Ca2+]i in (C) (open circles). The concentration dependence was described with a Hill equation yielding an apparent IC50 value of 106 ± 4 nM and a Hill coefficient of 4.2 ± 0.2 (n = 8–12). The binding of CaM to the GST–34 fusion protein was measured in GST pull-down assays. In (B), a Coomassie-stained gel shows the co-precipitated CaM at the level of the arrow for the indicated Ca2+ concentrations. The binding signal was integrated and plotted in (D) (filled circles) as a function of the Ca2+ concentration. Filled squares and triangles represent two more independent experiments. The continuous curve represents a data fit to a Hill equation with the parameters: KD = 480 ± 40 nM, Hill coefficient = 1.7 ± 0.2. (D) According to the data shown in (B), the binding of CaM to hEAG1 depends on Ca2+. This is not the case for the binding of CaM to the Ca2+-activated K+ channel hIK. The panels show [35S]CaM overlay assays with a C-terminal fragment of hIK [hIK: GST–hIK (286–427)] and the GST fusion protein GST–34 of hEAG1 in 1500 nM Ca2+ and in the absence of Ca2+. Download figure Download PowerPoint The weak binding of CaM at physiological Ca2+ concentrations suggests a regulatory mechanism mainly controlled by binding and release of the sensor molecule. This is in contrast to other Ca2+-regulated K+ channels from the SK/IK family, for which a constitutive binding of CaM has been proposed (Xia et al., 1998). For direct comparison of the CaM-binding properties of hEAG1 and a member of the KCa channels, we fused the complete C-terminus of the human IK channel (hIK or hSK4) to GST. Figure 5D shows a comparison of this construct with the GST–34 construct containing the binding motif of hEAG1 in an overlay assay. As expected, the hEAG1 fusion only bound CaM at high [Ca2+] but not in the absence of Ca2+. In contrast, the hIK fusion showed significant binding even in 10 mM EGTA, although a reduction compared with binding in 1500 nM Ca2+ was detectable. One calmodulin molecule closes the channel The fact that the IC50 found for hEAG1 channel inhibition by Ca2+/CaM is lower than the KD for CaM binding to the hEAG1 protein (Figure 5) suggests a closing mechanism that requires <4 CaM molecules bound to the channel tetramer. To address this question, we co-injected hEAG1 mRNA with mRNA coding for the C7 mutant into Xenopus oocytes and analysed the Ca2+ sensitivity of the resulting currents in inside-out patches. The quality of the two mRNA species was compared, measuring the total current obtained when three different mRNA concentrations for each channel type were injected into Xenopus oocytes. As illustrated in Figure 6A, both mRNAs resulted in approximately equal current amplitudes. Using constant amounts of mRNA for the Ca2+-insensitive mutant, we then set up a series of co-injections with decreasing content of wild-type mRNA in order to create channels with a reduced number of binding sites per tetramer. In inside-out patches from such oocytes, we first determined for each patch the maximum current amplitude in Ca2+-free solution. The current remaining after application of CaM in 200 nM Ca2+ was subsequently measured. The fractions of the Ca2+-insensitive currents were plotted over the fraction of wild-type subunits in the respective cell (Figure 6B). The calculation of the wild-type fraction is based on the assumption that both mRNA species express equally well and that the subunit assembly is not impaired by the mutation. The curves superimposed on the data in Figure 6B represent the theoretically expected sensitivities, based on a binomial distribution of the channel subunits among CaM-sensitive wild-type and CaM-insensitive C7 subunits (see Equation 1). The solid curve is the prediction of the assumption that only one bound CaM molecule is required to close the channel. The alternative assumptions, namely that either two, three or four bound CaM molecules are required to close a channel, do not describe the data well (dotted curves). Thus, the data clearly support the hypothesis that one CaM molecule bound to hEAG1 channels is sufficient for Ca2+-induced current inhibition. Figure 6.Stoichiometry of CaM–hEAG1 interactions. In order to address the question of how many CaM molecules are necessary to inhibit hEAG1 channels, we investigated the Ca2+/CaM sensitivity of oocytes injected with mixtures of mRNA coding for the wild-type and mutant C7. In (A), the expression levels measured with the two-electrode voltage clamp method at +20 mV for the two channel types are shown for different amounts of mRNA (n = 10–15), indicating that the expression levels in this situation are similar. (B) The Ca2+/CaM sensitivity was measured in 200 nM free Ca2+. The relative remaining current after Ca2+/CaM application was plotted as a function of the expected fraction of wild-type subunits (n = 4–6). The thick continuous line indicates the prediction of the Ca2+/CaM sensitivity assuming that one wild-type subunit is sufficient to confer Ca2+/CaM sensitivity to the channel complex. The dotted curves indicate the alternative predictions for the requirement for two, three or four wild-type subunits. To match the observed data to the model considering two CaM molecules per tetramer, we would have to assume that the wild-type expressed four times more strongly than the mutant. This is not supported by the data shown in (A). The fit to the other models would require an even more biased expression of the wild type, affording correction factors of 11 and 35, respectively. Download figure Download PowerPoint Discussion We have identified CaM as the Ca2+ sensor for human EAG K+ channels. CaM binds to the C-terminal domain of hEAG1 in a Ca2+-dependent manner. It thus does not form a constitutive subunit of the channel. Our studies demonstrate that the binding of one Ca2+/CaM complex molecule per channel tetramer is already inducing current inhibition. By means of this stoichiometry, EAG channel availability is regulated just above basal Ca2+ levels of resting cells; despite a binding KD for each individual subunit of ∼480 nM Ca2+, half-maximal channel inhibition is observed at ∼100 nM. Exactly the same Ca2+ dependence of inhibition was found for EAG channels in human melanoma cells (Meyer et al., 1999). This functional match, as well as the finding that a CaM-specific binding peptide inactivated the Ca2+-sensitive factor in cell lysates, strongly suggests that CaM is a binding partner of hEAG channels under in vivo conditions. A comparable Ca2+ regulation of K+ channels has thus far only been observed for KCa channels. These channels show no or only weak voltage dependence and are activated by elevated intracellular [Ca2+]. EAG channels, however, belong to the large group of voltage-activated K+ channels that exhibit delayed rectifier characteristics. Our results show that the functional principle of CaM-mediated ion channel regulation must be extended to delayed rectifier channels and indicate a unique function of EAG channels within this group. Interestingly, the same sensor protein that is known to activate KCa channels in response to rising [Ca2+] can reduce the availability of EAG channels. This reverse regulation can take place in the same cell, as both EAG and channels of the SK/IK type were identified in human melanoma cells (Meyer et al., 1999). This apparent paradox is explained by the different regulatory mechanisms in both systems. hEAG1 channels are closed by bindin