Individual Subunits Contribute Independently to Slow Gating of Bovine EAG Potassium Channels*

Roland SchönherrDagger , Solveig HehlDagger , Heinrich Terlau§, Arnd Baumann, and Stefan H. HeinemannDagger parallel

From the Dagger  Max-Planck-Gesellschaft, Arbeitsgruppe Molekulare und zelluläre Biophysik, Drackendorfer Strasse 1, D-07747 Jena, § Max-Planck-Institut für experimentelle Medizin, Abteilung Molekulare Biologie neuronaler Signale, Hermann-Rein-Strasse 3, D-37075 Göttingen, and  Institut für Biologische Informationsverarbeitung, Forschungszentrum Jülich, D-52425 Jülich, Germany

    ABSTRACT
Top
Abstract
Introduction
References

The bovine ether à go-go gene encodes a delayed rectifier potassium channel. In contrast to other delayed rectifiers, its activation kinetics is largely determined by the holding potential and the concentration of extracellular Mg2+, giving rise to slowly activating currents with a characteristic sigmoidal rising phase. Replacement of a single amino acid in the extracellular linker between transmembrane segments S3 and S4 (L322H) strongly reduced the prepulse dependence and accelerated activation by 1 order of magnitude. In addition, compared with the wild type, the half-activation voltage of this mutant was shifted by more than 30 mV to more negative potentials. We used dimeric and tetrameric constructs of the bovine eag1 gene to analyze channels with defined stoichiometry of mutated and wild-type subunits within the tetrameric channel complexes. With increasing numbers of mutated subunits, the channel activation was progressively accelerated, and the sigmoidicity of the current traces was reduced. Based on a quantitative analysis, we show that the slow gating, typical for EAG channels, is mediated by independent conformational transitions of individual subunits, which gain their voltage dependence from the S4 segment. At a given voltage, external Mg2+ increases the probability of a channel subunit to be in the slowly activating conformation, whereas mutation L322H strongly reduces this probability.

    INTRODUCTION
Top
Abstract
Introduction
References

Voltage-gated potassium channels (K+ channels) play major roles in the control of cellular resting potentials, and they are involved in the regulation of firing rate and shaping of action potentials in excitable cells (1). The progress of molecular cloning over the past 10 years revealed an astonishing diversity within this group of membrane proteins, which comprises several related gene families (2). The ether à go-go gene, which has originally been cloned from Drosophila, provided the name for a growing sub-family of K+ channels that contain six transmembrane segments (S1-S6) per subunit and a binding motif for cyclic nucleotides in the cytoplasmic carboxyl terminus of each subunit. At present this subfamily consists of three members, the eag gene, the eag-like K+ channel gene (elk), and the eag-related gene (erg) (3). Further diversity is caused by splice variants or isoforms that have been described for all three genes (4-7). Whereas the ERG channel is responsible for the cardiac IKr current (8), the physiological role of EAG channels still remains to be identified. Recently the first examples of native EAG currents have been described. A human neuroblastoma cell line was found to express human eag in a cell cycle-dependent manner (9). In human myoblasts, the expression of human eag is correlated with a non-inactivating delayed rectifier K+ current, which is involved in myoblast fusion (10).

A characteristic feature of all EAG channels that have been examined so far is the unusual kinetics of the activation process. It displays a strong dependence on the prepulse potential (11), reminiscent of the shift originally described by Cole and Moore (12). Test pulses from potentials just below the activation threshold (e.g. -70 mV) lead to rapid activation, whereas hyperpolarizing prepulses result in very slow activation kinetics. Terlau et al. (13) showed that the slow activation of rat EAG not only occurs at unphysiologically low holding potentials but also under physiological conditions in the presence of extracellular Mg2+ at mM concentrations. The unusual gating of EAG channels was described using a sequential gating model that considered two voltage-dependent transitions for each single subunit (13). The underlying molecular mechanism, however, has not yet been elucidated.

In this paper we describe the electrical properties of a point mutation introduced into the bovine eag1 gene. The mutation in the extracellular linker of transmembrane segments S3 and S4 strongly decreased both, the Cole-Moore shift of b-EAG1 activation and the apparent regulation by external Mg2+. We used this mutation as a tool to separate slow and fast gating processes. To control the stoichiometry within the channel tetramers, we constructed dimeric and tetrameric b-eag1 genes with different numbers and orders of the mutated subunits. Using this approach, we could dissect the complex conformational changes in the multimeric channel protein and assign them to gating transitions of individual subunits. The activation kinetics of the linked constructs could be described by a fully independent model, assuming a binomial distribution of the individual subunits between two closed states and a voltage-dependent slow transition between these conformations. The wild-type subunits, participating in a heterotetrameric channel, are decisive for the slow gating kinetics.

    EXPERIMENTAL PROCEDURES

Construction of Channel Mutants and mRNA Synthesis-- The bovine eag1 gene (7) was used to generate the mutants and chimeric constructs. Constructs were subcloned into the oocyte expression vector pGEMHE (14) using the restriction sites BamHI and HindIII. The point mutation L322H was created by site-directed mutagenesis using the polymerase chain reaction. The polymerase chain reaction product was transferred into the wild-type plasmid as a BstBI-MluI fragment, and the sequence of the complete fragment was verified by cycle sequencing of the plasmid DNA. For construction of dimeric and tetrameric eag genes, we introduced EcoRV and HindIII restriction sites at the 3' end of the eag gene. The modified 3' end reads GGCGCAAGCggatatcaatgataagctt (lowercase letters are for added nucleotides). This construct was opened with EcoRV and HindIII, and a second gene copy was coupled to the 3' end as an SmaI-HindIII fragment. The SmaI site is part of the upstream region of b-eag in pGEMHE (cccggggatccaccATG, start codon is in capitals). The junction of the two gene copies encodes five additional amino acids (GWGST). Two dimer constructs were then linked using the same restriction sites to obtain a tetrameric gene. The T7 mMessage mMachine Kit (Ambion, Austin, TX) was used for mRNA synthesis after linearization with NheI.

Electrophysiological Measurements-- Stage V oocytes were prepared from Xenopus laevis as described previously (15), and 50 nl of in vitro transcribed mRNA (5-500 ng/µl) were injected. Currents were recorded at 20-23 °C in the period ranging from 1 to 7 days after injection. A two-electrode voltage clamp amplifier (Turbo-TEC 10CD, NPI electronic, Tamm, Germany) was used, and electrodes filled with 2 M KCl had resistances between 0.5 and 0.9 megaohms. The bath solution was normal frog Ringer's (NFR), containing 115 mM NaCl, 2.5 mM KCl, 1.8 mM CaCl2, 10 mM HEPES, pH 7.2 (NaOH). MgCl2 or NiCl2 was added to this solution at concentrations indicated for individual experiments. Thus, the external solution always contained 1.8 mM Ca2+ ions, as complete removal of divalent ions resulted in the emergence of a leak current without strong reduction of the Cole-Moore shift of b-EAG1 (not shown). Experiment control including pulse generation and data recording was performed with the Pulse + PulseFit software package (HEKA elektronik, Lambrecht, Germany). A P/n method, supported by Pulse + PulseFit, was used in all experiments for correction of leak and capacitive currents.

Data Analysis and Modeling-- Data were analyzed using PulseFit, PulseTools (HEKA elektronik) and IgorPro (WaveMetrics, Lake Oswego, Oregon) running on Macintosh and PC-compatible computers. Analyzed parameters are presented as mean ±S.E., with n = number of independent experiments.

Normalized conductances as shown in Fig. 1C were obtained by fitting the following equation to the current-voltage data:
I(V)=&Ggr;V<FR><NU>1−e<SUP><UP>−</UP>(V<UP>−</UP>E<SUB><UP>rev</UP></SUB>)/25<UP>mV</UP></SUP></NU><DE>1−e<SUP><UP>−</UP>V/25<UP>mV</UP></SUP></DE></FR> <FR><NU>1</NU><DE>1+e<SUP><UP>−</UP>(V<UP>−</UP>V<SUB>h</SUB>)/k</SUP></DE></FR> (Eq. 1)
where Gamma  is the maximal conductance, and Erev is the estimated reversal potential. The first term accounts for the single-channel conductance according to the Goldman-Hodgkin-Katz equation. The last term describes the open probability (popen) of the channels, characterized by the voltage of half-maximal activation, Vh, and a slope factor, k.

Time course of activation of wild-type b-EAG channels was described using the following equation:
I(t)=a<FENCE>(1−p)<SUP>n</SUP>(1−e<SUP><UP>−</UP>t · k<SUB>f</SUB></SUP>)+<FENCE><LIM><OP>∑</OP><LL>i<UP>=</UP>1</LL><UL>n</UL></LIM><FENCE><AR><R><C>i</C></R><R><C>n</C></R></AR></FENCE>p<SUP>i</SUP>(1−p)<SUP>n<UP>−</UP>i</SUP>(1−e<SUP><UP>−</UP>t · k<SUB>s</SUB></SUP>)<SUP>i</SUP></FENCE></FENCE> (Eq. 2)
In this equation, the symbols have the following meaning: a = amplitude, kf = rate constant of channel activation when all slow gates are activated, ks = rate constant of activation per slow subunit, n = number of slow subunits, p = probability for a subunit being in a state from which activation is slow. In the figures, the speed of activation is shown as time constant, i.e. the inverse of the rate constants (e.g. tau s = 1/ks). To account for the limited clamp speed of the two-electrode voltage clamp, a time delay of 1 ms was assumed. This model implies a binomial distribution of subunits between "fast" and "slow" states. Activation of channel constructs with only two or one wild-type subunit were described by this equation, assuming n = 2 or 1, respectively. In this case p denotes the probability of being in a "slow" state for wild-type subunits only. As shown in the results section, this equation gives a very reliable description of the data obtained in elevated concentrations of divalent cations (e.g. Mg2+) and using a negative holding potential. In such cases (p > 0.5), the first term, describing fast activation, basically vanishes ((1 - 0.5)4 = 0.0625). Thus, the activation is described by only three free parameters: a, p, and ks. In cases where p is small, the fast component adds another free parameter, kf. We chose to describe fast activation with a single-exponential component rather than with the fourth power, because the limited speed of the two-electrode voltage clamp and the rapid activation rate, kf, hampered a faithful resolution. The results obtained for the slow Mg2+-dependent gating of b-EAG channels, however, were not affected by this limitation.

Individual data traces were fitted according to Equation 2 using a Levenberg-Marquardt algorithm as implemented in IgorPro. Whole sets of data traces similar to those shown in Fig. 5 were simultaneously fitted according to Equation 2 using a Simplex algorithm, programmed after Caceci and Cacheris (16). In this case, identical kf and ks values were used for all traces, whereas individual p values were assigned to each trace as p is a function of prepulse potential (see Fig. 5).

    RESULTS

Mutation L322H Shifts the Voltage Dependence of Activation to Hyperpolarized Voltages-- We were interested in identifying amino acid residues that influence the characteristic gating of EAG channels. As shown previously, deletions in the cytoplasmic amino terminus of rat EAG affect both voltage dependence and activation kinetics of the channels (17). A point mutation at the cytoplasmic face of the S4 segment (H343R) was found to largely compensate for the effect of such deletions (17), and the same phenotype was found for the analogous mutations introduced into the bovine EAG channel (not shown). Recently, Tang and Papazian (18) described mutations close to the extracellular part of the S4 segment of the Drosophila EAG channel. A point mutation that replaced a leucine residue in position 342 by histidine led to a shift of the current-voltage relation by 16 mV to depolarized voltages. We introduced the equivalent mutation at the corresponding position of b-EAG1 (L322H) (Fig. 1A). Under a two-electrode voltage clamp, this mutant gave rise to delayed rectifier currents, very similar to wild-type currents when expressed in Xenopus oocytes (Fig. 1B). A comparison of popen over the tested voltage range shows that channels carrying the mutation L322H open at more negative voltages than wild-type channels (Fig. 1C). The voltage required for half-maximal activation (Vh) of the mutant channels was shifted by more than 30 mV to hyperpolarized voltages (wild type, -11.8 ± 2.2 mV, n = 8; L322H, -45.1 ± 1.8 mV, n = 12), whereas the slope factors (k) were not significantly different (Fig. 1D).


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Fig. 1.   Mutation L322H induces a shift in the voltage-dependent activation of b-EAG1. A, amino acid alignment of the putative S3 and S4 transmembrane segments of b-EAG1 and d-EAG channels. In the d-EAG sequence, only, deviations from b-EAG1 are shown, and identical residues are indicated by hyphens. The arrow indicates the leucine residue that has been replaced by histidine in b-EAG1 (L322H) in analogy to the d-EAG mutation (L342H) described by Tang and Papazian (18). B, currents were measured with a two-electrode voltage clamp from oocytes injected with mRNA encoding wild type b-EAG1 (top) or the mutant L322H (bottom). C, the current magnitude was measured in the last 10% of the depolarization segments and plotted as a function of voltage. The current was described by Equation 1. The open probability is given by a single-component Boltzmann function characterized by the half-maximal activation voltage, Vh, and the slope factor, k. The single-channel current was accounted for by assuming a single-channel conductance according to the Goldman-Hodgkin-Katz formalism with a reversal potential of -90 mV (Equation 1). Data were fitted to voltage values ranging between -80 and -20 mV (L322H, filled circles) or +10 mV (wild type, open circles) to avoid the influence of current rectification at higher potentials; data points that were not used for the fit are shown as small squares. Data sets from individual oocytes were then transformed to popen values using the data fit results. These popen values were compiled and are plotted in C as mean ±S.E. for the wild type (n = 8) and the mutant L322H (n = 12). Data points are connected by straight lines. In D, the resulting Vh and k values are compared using box plots. Bath solution: NFR.

Mutation L322H Accelerates Activation Kinetics-- To characterize the activation kinetics of b-EAG1 and the effect of the L322H mutation in more detail, we applied repeated test pulses to +40 mV and varied the prepulse potential between -130 and -70 mV with increments of 10 mV. The resulting current traces, recorded under a two-electrode voltage clamp are shown in Fig. 2A. As extracellular bath solution, we used NFR, containing different concentrations of either Mg2+ or Ni2+ ions. b-EAG1 channels responded to changes in the holding potential in a very similar fashion as described previously for r-EAG channels (13). Hyperpolarizing prepulses caused a marked slowing of current activation, even in 0 mM [Mg2+]o. Increasing the concentration of extracellular Mg2+ to 10 mM resulted in additional slowing of the activation kinetics. Further slowing of the activation was observed in solutions containing 1 mM Ni2+. For a model-independent description of the time course of activation, we plotted the time to reach 80% of the maximum current amplitude as a function of the prepulse potential (Fig. 2B). In 0 mM [Mg2+]o, the slowing effect of the prepulses on b-EAG1 saturates at about -130 mV (open circles), whereas in 2 mM [Mg2+]o saturation already occurs at potentials more negative than -90 mV (open triangles). In 10 mM [Mg2+]o, the channels always activated slowly, almost independent of the prepulse voltages (open squares). For comparison, the mutant channels, analyzed under the same ionic conditions, showed that the point mutation L322H accelerated the activation kinetics by about 1 order of magnitude (closed circles). In addition, the prepulse dependence in Mg2+-free NFR solution was almost completely abolished. The mutant channels still responded to variations in the extracellular cation concentration. In 10 mM [Mg2+]o, the time to reach 80% activation at different holding potentials is very similar to the values found for the wild type in the absence of Mg2+ (compare open circles and filled squares in Fig. 2B).


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Fig. 2.   Mutant L322H strongly reduces the prepulse dependence of activation. A, activation of wild-type (left) and L322H (right) channels was assessed by application of depolarizations to +40 mV from various holding potentials. The bath solution (NFR) contained, in addition to 1.8 mM Ca2+, the indicated divalent cations. Although activation of the wild-type channels shows the typical dependence on both, holding potential and divalent cations, activation of L322H channels is generally faster. The speed of activation was quantified by measuring the time required for the current to reach the 80% level. This is plotted in B as a function of voltage (n = 4-6). The use of the symbols is indicated in A. The data points were connected by straight lines. Current traces in A were normalized to the current level at the end of the depolarizations (depolarizations of 5 s were applied for wild-type channels in Ni2+ to obtain a steady-state level for normalization (not shown)).

Quantitative Description of b-EAG1 Gating and Mg2+ Regulation-- For a quantitative comparison of the gating kinetics of mutant and wild-type channels, we built on a gating model introduced by Terlau et al. (13). According to that model, EAG channels undergo two qualitatively different classes of conformational transitions upon depolarization. Rapid activation steps follow a slow conformational change, which is dependent on extracellular divalent cations. The latter steps are similar to what has been described for other voltage-gated potassium channels. Therefore, we assumed at the holding potential, a binomial distribution of all four individual subunits between the slow gates being turned on or off. The probability of being in a "slow" state (p) will be a function of both the holding potential and the concentration of extracellular divalent cations. Starting from such a steady-state distribution among slow and fast states, the activation time course was calculated by assuming single-exponential relaxation of all "slow" subunits with a slow rate constant, ks. Fast subunits contribute to the lumped rapid activation according to kf (Equation 2). Fits according to this simple model (Equation 2 with n = 4) matched perfectly to experimental data from b-EAG1, obtained at various concentrations of extracellular Mg2+ (Fig. 3A, left, fits in black), regardless of whether prepulses to -130 mV or to -70 mV were applied. The current traces shown represent the mean currents of six independent experiments. Individual traces from different experiments were first normalized to the maximum current level at the end of the test pulse. These traces were then used to calculate mean current traces. The employed model is obviously well suited to describe both the hyperpolarization-induced shift and its regulation by Mg2+. The composition of the mean current from contributions of fast and slow gating channels is well demonstrated in 10 mM Mg2+ using a prepulse voltage of -70 mV (Fig. 3A, lower left panel, lowest trace). A rapidly activating current component is followed by a slowly activating component. The relative amplitude of the fast component with about 15% of the total current is in good accordance with a probability of about 38% for individual subunits to go through the slow gating transition. The two free fit parameters, namely the probability of starting from a slow state, p, and the time constant for a slow transition, 1/ks, are plotted as a function of [Mg2+] in Fig. 3, B and C, respectively (open symbols). Although p is a function of both, the prepulse potential and [Mg2+], 1/ks is only a function of [Mg2+], thus providing a test for the validity of this simple approach.


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Fig. 3.   Quantitative description of the activation in various Mg2+ concentrations. A, activation elicited by voltage steps to +20 mV were measured for the wild type (open symbols) and L322H (closed symbols) from holding potentials of -70 (bottom) and -130 mV (top). The extracellular Mg2+ concentrations were: 0.2, 0.5, 1, 2, 5, and 10 mM. Data traces from 6 oocytes were normalized to the current level after 1-s depolarization. The mean traces are shown in A. A data fit was performed according to Equation 2 using n = 4, and the fit results (black) are superimposed to the data (gray). In the left panels, the fit matches perfectly such that it cannot be distinguished from the data. The two free parameters of Equation 2, namely the probability of starting in a slow state (p-slow) (B) and the time constant of activating a subunit from a slow state (1/ks) (C), are plotted as a function of the Mg2+ concentration. Data points are connected by straight lines. Symbols as indicated in A.

Data for the mutant L322H (Fig. 3A, right) were analyzed with the same method, using n = 4 in Equation 2. Because of the more rapid activation, fits were only performed to the first 300 ms after depolarization. Although the initial part of the traces was described well, a minor third current component with very slow kinetics could not be accounted for. The fit parameters for the mutant are shown as closed symbols in Fig. 3, B and C. p was generally much smaller than for the wild type, and the time constants for the activation of the slow gates did not depend on [Mg2+]. In addition, this time constant was similar to the time constant of wild-type subunits in the absence of Mg2+ (approx 50 ms). Thus, these results indicate that data of the wild-type channel were adequately described by Equation 2. Moreover, the mutant subunits do not seem to contribute significantly to the slow gating, and they can be safely discriminated from wild-type subunits in high [Mg2+].

Activation of Linked Channel Constructs-- Potassium channels are formed as symmetric tetramers of alpha -subunits assembled around a central pore (19-21). Thus, the S3-S4 linker region, which is apparently involved in the process leading to slow activation, is represented in four copies in the functional channel. This raises the question, whether the slow activation of wild-type channels after hyperpolarization is a concerted action of all four subunits (mediated by the binding of up to four Mg2+ ions) or if it is a function of single and independent gating events. To assess this problem, we chose the approach of concatenated genes to control the stoichiometry of fast (L322H) and slow (wild type) gating subunits in the tetramer. A dimeric construct containing one mutant and one wild-type subunit was made by linking the cytoplasmic carboxyl terminus of a mutant to the amino terminus of a wild-type subunit (m-w, see "Experimental Procedures"). Expression of this construct should result in channels with a 2 + 2 stoichiometry. The same stoichiometry was expected for another dimer (w-m) and two tetrameric constructs (m-w-w-m, w-m-w-m), which resulted from the coupling of two dimers. All constructs were functionally expressed in oocytes. To obtain similar current amplitudes, mRNA concentrations 5 times and 20 times higher than for the wild type had to be injected for dimers and tetramers, respectively. A channel formed by wild-type dimers (w-w) did not show significantly different activation than channels formed by wild-type monomers (not shown). As shown in Fig. 4A, all four constructs with a 2 + 2 stoichiometry resulted in intermediate activation kinetics when compared with wild-type (w-w) or mutant (m) channels. An additional tetramer construct containing only one wild-type subunit (m-m-m-w) activated faster than channels with a 2 + 2 stoichiometry but slower than the mutant monomers. In the upper panel of Fig. 4A, currents were elicited from hyperpolarized holding potentials in the presence of 5 mM Mg2+, favoring the slow channel activation. In the lower panel,we applied test pulses from a holding potential of -70 mV. All traces shown are mean traces from at least 5 individual experiments. Note that the four tested constructs with theoretical 2 + 2 stoichiometry show similar but not identical activation kinetics. This probably reflects a possible deviation of the actual channel assembly from the expected structures, as depicted in Fig. 4B (see "Discussion"). Apart from these minor deviations, the linked constructs clearly show that the activation kinetics of b-EAG channels is determined by the gating properties of the individual subunits constituting the channel rather than by a concerted action that requires all four subunits for slow activation.


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Fig. 4.   Activation of linked constructs. A, activation time courses of the indicated channel constructs were measured in 5 mM Mg2+. Voltage steps from the indicated holding potentials were elicited to +20 mV. The data traces are mean traces from 5-10 individual experiments, normalized to the maximal current. It is clearly seen that an increasing number of wild-type subunits per channel results in a decreased speed of activation. B, schematic for the ideal assembly of the constructs to form functional potassium channels.

In contrast to this additive regulation of the gating kinetics, the changes in the current-voltage dependence of steady-state currents were much more dominated by wild-type subunits. The Vh values of the tetramers with 2+2 stoichiometry (m-w-w-m: -11.1 ± 0.7 mV, n = 11; w-m-w-m: -6.1 ± 2.3 mV, n = 7) did not significantly differ from the wild type (-11.8 ± 2.2 mV, n = 8). Only the m-m-m-w tetramer was shifted to hyperpolarized potential (-22.9 ± 0.6 mV, n = 12), though much less pronounced than the completely mutated channel (-45.1 ± 1.8 mV, n = 12). Obviously the wild-type subunit, which needs more positive potentials to gate open, dominates the behavior of the channel.

Quantitative Analysis of Linked Channel Constructs-- The data shown in Fig. 4 already indicate that an increasing number of wild-type subunits per channel progressively slow down channel activation. As the mutated subunits give rise to considerable faster activation and small p values (see Fig. 3), it might be possible to describe the data for the linked constructs with a 2 + 2 or 1 + 3 stoichiometry by using Equation 2 with n = 2 or 1, respectively. In this case, the mutant subunits are only considered to contribute to the lumped fast activation component. In addition, it should be assumed that both rate constants for activation, kf and ks, are only a function of the test potential and not of the prepulse potential. Mean normalized currents in 5 mM Mg2+, elicited by test pulses to +20 mV, are shown in Fig. 5 for the indicated constructs and prepulse voltages. All recorded currents of the wild-type construct (w-w) were simultaneously fitted with Equation 2 with n = 4, yielding 1/kf = 10 ms and 1/ks = 215 ms. Note that in contrast to the fits shown in Fig. 3, we now used more constrained fit conditions, as nine different activation time courses (corresponding to 9 prepulse potentials) had to be described simultaneously. Fitted curves are superimposed (black), indicating that even this heavily constrained fit describes the data (gray) well. The resulting p values are plotted in Fig. 6 as a function of prepulse potential (open circles). The data of constructs m-w and m-m-m-w were then fitted to Equation 2 assuming n = 2 or 1, respectively. The data fits resulted in 1/kf = 9.9 ms and 1/ks = 152 ms for m-w and 1/kf = 11.9 ms and 1/ks = 171 ms for m-m-m-w. It is clearly shown that the data are described well. Similar fits with n = 4 gave consistently worse results (not shown). The p values are also shown in Fig. 6, indicating that, similarly to the ks and kf values, they are rather similar for wild-type subunits in a homotetrameric channel (w-w). Mutant subunits, however, yield much smaller p values, as already shown in Fig. 3 and smaller time constants: 1/kf = 7.2 ms and 1/ks = 79 ms (mutants (m) were fitted with Equation 2 using n = 4). The shown fits and the high similarity of 1/ks, 1/kf, and p-slow values for the wild-type subunits in all analyzed constructs strongly support our assumption that slow gating of EAG channels can be quantitatively described by the independent gating of the participating wild-type subunits.


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Fig. 5.   Superposition of normalized and averaged data traces (gray) obtained in 5 mM Mg2+ for voltage steps to +20 mV from the indicated holding potentials. The data were obtained from oocytes expressing the indicated channel constructs: w-w (wild-type dimer, n = 4), m-w (L322H, wild type, n = 9), m-m-m-w (tetramer with three L322H subunits and one wild-type subunit, n = 5), and m (L322H, n = 5). Superimposed to the data are the results of simultaneous data fits (black). The theory of these fits considers the number of wild-type subunits per channel (for details see text).


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Fig. 6.   The p-slow values of the wild-type subunits as a result of the data fits shown in Fig. 5 are shown here as a function of the holding voltage. For the monomeric mutant channels, activation was described like that of the wild type, yielding estimates for the p-slow values of mutant subunits. Because these are much smaller than those of the wild type, they were neglected for the theoretical description of the heteromeric constructs. Data points for the mutant (m) were connected by straight lines, and the other data points are superimposed to Boltzmann fit functions (see "Discussion").


    DISCUSSION

EAG potassium channels activate in a rather unique way as the kinetics of channel opening strongly depend on the voltage that the channel proteins experience before depolarization. Hyperpolarizing prepulses result in a slowed activation and cause a more sigmoidal rising phase of the current waveform (9, 11, 13, 22). Similar to hyperpolarizing prepulses, extracellular divalent cations, e.g. Mg2+, slow the activation kinetics and increase the sigmoidicity of the rising phase. Because of this similarity, a common mechanism has been proposed for Cole-Moore shift and Mg2+ regulation. Mg2+ ions could shift the equilibrium voltage between two closed states, leading to either slow or fast activation (13). Detailed analysis of the prepulse dependence and the effect of extracellular Mg2+ gave rise to a model in which up to four slow gating steps precede fast channel activation (13).

Importance of the S3-S4 Linker for Cole-Moore Shift and Mg2+ Regulation-- In Drosophila EAG, (d-EAG) channels, Tang and Papazian (18) found that deletions and point mutations in the extracellular S3-S4 linker had severe effects on the gating characteristics. A deletion of five charged residues slowed the activation in the presence of Mg2+ ions. A direct hint, pointing to the S3-S4 linker as regulator of the Cole-Moore shift, was the observation that a naturally occurring splice variant (b-EAG2) of the recently cloned bovine EAG channel is characterized by a 27-amino acid insertion in this linker, making this protein more sensitive to [Mg2+]o compared with b-EAG1 (7). Mutagenesis in b-eag1 now provided further evidence for the importance of the S3-S4 linker. The single amino acid exchange in the b-EAG1 channel (L322H) significantly reduced the prepulse dependence of the channels (Fig. 2). Interestingly, in addition to the faster activation, the voltage dependence of the steady-state current was shifted toward hyperpolarizing voltages for this mutant (Fig. 1). This is in striking contrast to a depolarizing shift observed by Tang and Papazian (18) for the d-EAG L342H mutant. The opposite effects of analogous mutations may be because of further differences in the S3-S4 linker regions of both channels (Fig. 1A).

The Mutation L322H Reduces Cole-Moore Shift in b-EAG1-- The mutation L322H caused a strong reduction of the EAG-specific slow activation process. In particular, the prepulse dependence and the influence of extracellular divalent cations was strongly reduced. It could be speculated that L322H alters a regulatory Mg2+ binding site on the channel and thereby leads to faster channel activation. However, this interpretation would not explain the accelerated activation kinetics of L322H channels even in the absence of Mg2+. Furthermore, a leucine is very unlikely to participate directly in binding divalent cations. Thus, it appears that the mutation primarily reduces the marked prepulse dependence of the channels but does not abolish the binding of Mg2+ ions.

An Independent Gating Model Describes the Cole-Moore Shift-- For a quantitative description of the gating characteristics of wild-type and mutant channels, we assumed a binomial distribution of four subunits among slow and fast states. This simple independent model yielded convincing fits to the experimental data, suggesting that the main effect of the L322H mutation is a strong reduction of the probability for a subunit to be in the slowly activating state (p-slow), both in the absence and presence of Mg2+ ions. One could speculate that Mg2+ binding is reduced in the fast state, but there is no direct evidence yet. Interestingly, the time constant of L322H subunit activation does not depend on the Mg2+ concentration anymore, whereas for the wild-type channels, it is increased by a factor of 2 in 10 mM [Mg2+] (Fig. 3C). Presently, it is not clear whether or not this results from limitations of the simple model employed. It also remains to be investigated to what extent changes in surface potential with increasing [Mg2+] can account for such an effect. In this respect it is noteworthy that an analysis of b-EAG1 activation with double exponentials, thus not taking into account an independent activation of up to four subunits, resulted in ks values independent of [Mg2+] (7). Our previous results with rat EAG (13) and the results with b-EAG1, strongly suggest that the independent model gives rise to better fits without introducing more free parameters.

Concatenated Gene Constructs of b-eag1 Show Independent Subunit Gating-- As our independent model for the slow gating of b-EAG channels was well suited to fit the ionic currents, the Cole-Moore shift of the channels should be because of a voltage-dependent gating step that is independent for all four subunits. To assess this question experimentally, we engineered channel constructs of concatenated wild-type and mutant eag1 genes, leading to dimers and tetramers with various combinations of the different subunits. The aim was to control the number of subunits in the channel that undergo the slow gating transition. Mutant L322H was well suited for that purpose as its rapid gating property can be safely distinguished from wild type. Although all combinations gave rise to functional channels, the expression level decreased with the increasing number of linked subunits. Although concatenated genes became a common tool to assess subunit stoichiometry (14, 20, 23-28), the properties of such channels cannot necessarily be explained from the expected assembly of the constructs (23, 24). Using dimeric constructs of Shaker K+ channels, McCormack and co-workers (23) described a preferential insertion of the NH2-terminal subunit into the tetramers. Thus, it may happen that functional tetramers are formed from three or even four dimers if only one-half of a dimer contributes to the channel. Therefore, quantitative analysis of linked constructs should include controls with opposite order of the linked subunits. We constructed m-w and w-m dimers and found that activation of w-m dimers was slightly slower than that of m-w dimers (Fig. 4A), suggesting a smaller probability of functional incorporation of the carboxyl-terminal subunit. The two tetrameric constructs with 2 + 2 stoichiometry showed almost no deviations in the activation kinetics, regardless of the order of the subunits (m-w-m-w or m-w-w-m), indicating the assembly of the complete entity. However, all tetramers lead to lower current amplitudes, probably because of impaired translation/translocation processes for these large proteins. In summary, we found that linked b-EAG1 channels do form functional complexes that possess almost the expected configuration as illustrated in Fig. 4B.

Linked Channel Constructs Support Independence of the Slow Gating Transitions-- After validating the approach of concatemer expression of b-EAG1 with L322H mutations, we set out to perform a quantitative description of channel activation, based on the predicted subunit stoichiometry. As shown in Fig. 5, simultaneous data fits to whole Cole-Moore data sets resulted in reasonable fits of the wild type and constructs with a 2 + 2 and 1 + 3 subunit stoichiometry. We chose the tetramer m-m-m-w; given the slightly smaller probability of functional incorporation of the last subunit, any slow activation will be a result of functional constructs of exactly that stoichiometry. Thus, these results show that the slow, Mg2+-dependent activation of b-EAG channels is mediated by the slow activation of each of the four subunits. At present, we cannot completely exclude that there is a certain degree of cooperativity among the slow gating steps. We tried to address this question by comparing the constructs m-w-m-w and m-w-w-m. As illustrated in Fig. 4B, upon correct assembly, these constructs both will have a 2 + 2 stoichiometry. In the latter case, channels contain two wild-type and mutant subunits as direct neighbors, whereas in the former case, the subunits are distributed diametrically. Comparison of the activation kinetics in 5 mM Mg2+ from -130 and -70 mV did not reveal differences between these isoforms. This suggests that the subunit position within the channel construct is not important for channel gating. The number of wild-type subunits per channel, however, may still give rise to cooperative gating. In the framework of the employed gating model (Equation 2), the dependence of p for wild-type subunits on the prepulse voltage should not depend on the construct type. As shown in Fig. 6, this is qualitatively the case when compared with mutant subunits. However, it seems that the steepness of the prepulse dependence depends on the number of wild-type subunits per channel. Boltzmann fits to the data plotted in Fig. 6 reveal slope factors of 12.6 mV for the wild type (w-w), 18.5 mV for the dimeric construct m-w, i.e. a 2 + 2 stoichiometry, and 28.2 mV for the tetrameric construct m-m-m-w, i.e. a 1 + 3 stoichiometry. This behavior may indeed be indicative of some subunit cooperativity, which remains to be investigated in more detail.

What can we learn from b-EAG1 L322H about the mechanism underlying the Cole-Moore shift of EAG channels? The introduced mutation is located close to the external part of the S4 segment. As it strongly reduced the prepulse dependence rather than the Mg2+ regulation, it is suggested that the S4 segment is involved in the conformational change underlying the slow gating of EAG channels. Thus, it is very likely that this conformational change also derives its voltage dependence from the S4 voltage sensor, although operating at a much more negative voltage than the fast gating, which leads to channel opening. This interpretation is consistent with our previous results, which showed that the effect of the NH2 terminus on the Cole-Moore shift is based on a close contact between this cytoplasmic domain and the cytoplasmic part of the S4 segment (17).

    ACKNOWLEDGEMENTS

We thank A. Robeta ner and S. Arend for excellent technical assistance and J. Tytgat for providing the vector pGEMHE.

    FOOTNOTES

* This work was partially supported by the DFG (SFB197).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

parallel To whom correspondence should be addressed: Tel.: ++49-3641-304541; Fax: ++49-3641-304542; E-mail: ite{at}rz.uni-jena.de.

    ABBREVIATIONS

The abbreviations used are: b-EAG, bovine EAG; d-EAG, Drosophila EAG; NFR, normal frog Ringer's solution.

    REFERENCES
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Abstract
Introduction
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