Solution Structure and Function of the "Tandem Inactivation Domain" of the Neuronal A-type Potassium Channel Kv1.4*,

Ralph WissmannDagger , Wolfgang BildlDagger , Dominik OliverDagger , Michael Beyermann§, Hans-Robert Kalbitzer, Detlef BentropDagger , and Bernd FaklerDagger ||

From the Dagger  Department of Physiology II, University of Freiburg, Hermann-Herder-Strasse 7, 79104 Freiburg, Germany, § Forschungsinstitut für Molekulare Pharmakologie, Campus Berlin-Buch, Robert-Rössle-Strasse 10, 13125 Berlin, Germany, and  Department of Biophysics and Physical Biochemistry, University of Regensburg, Universitätsstrasse 31, 93053 Regensburg, Germany

Received for publication, October 4, 2002, and in revised form, February 14, 2003

    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
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Cumulative inactivation of voltage-gated (Kv) K+ channels shapes the presynaptic action potential and determines timing and strength of synaptic transmission. Kv1.4 channels exhibit rapid "ball-and-chain"-type inactivation gating. Different from all other Kvalpha subunits, Kv1.4 harbors two inactivation domains at its N terminus. Here we report the solution structure and function of this "tandem inactivation domain" using NMR spectroscopy and patch clamp recordings. Inactivation domain 1 (ID1, residues 1-38) consists of a flexible N terminus anchored at a 5-turn helix, whereas ID2 (residues 40-50) is a 2.5-turn helix made up of small hydrophobic amino acids. Functional analysis suggests that only ID1 may work as a pore-occluding ball domain, whereas ID2 most likely acts as a "docking domain" that attaches ID1 to the cytoplasmic face of the channel. Deletion of ID2 slows inactivation considerably and largely impairs cumulative inactivation. Together, the concerted action of ID1 and ID2 may promote rapid inactivation of Kv1.4 that is crucial for the channel function in short term plasticity.

    INTRODUCTION
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INTRODUCTION
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Fast inactivation of voltage-gated K+ (Kv)1 channels is a major determinant for signal propagation and synaptic transmission in central nervous system neurons (1-4). Thus, A-type K+ channels were shown to regulate orthograde and retrograde propagation of dendritic action potentials (APs) in CA1 pyramidal neurons and to dynamically control presynaptic Ca2+ inflow into hippocampal mossy fiber boutons (2, 3). In the latter, repetitive stimulation (frequencies of >= 10 Hz) led to a broadening of the APs because of cumulative inactivation of A-type K+ channels. The prolonged AP resulted in an increased Ca2+ inflow, which in turn promoted an increased transmitter release and a consecutive potentiation of the evoked excitatory postsynaptic currents (2).

The molecular determinant of the rapidly inactivating K+ current in mossy fiber boutons most probably is Kv1.4, a Kv alpha -subunit with prototypic ball-and-chain-type inactivation (2, 4, 5). This classical gating process is brought about by a "plug-in" mechanism in which a protein domain termed "inactivation ball" occludes the ion pathway through interaction with a receptor site in the pore of the channel (6). Structure-function analysis showed: (i) that inactivation domains (ID) are formed by the first 20-40 amino acids at the N terminus of certain Kvalpha (7-10) and Kvbeta subunits (11, 12); (ii) that IDs inactivate channels independent of whether they are tethered to the protein or offered to the channel as a synthetic peptide (8, 9, 13, 14); (iii) that IDs enter the channel pore and compete with open pore blockers such as tetraethylammonium (TEA) (11, 15-17); and finally (iv) that NMR-derived solution structures of the known IDs vary from a well ordered and compact folding as in Kv3.4 ID (18) to the disordered and highly flexible ID from ShakerB (19).

Different from other alpha /beta subunits that harbor one ID in their N terminus, Kv1.4 was found to be equipped with two IDs with the ability to endow the channel with rapid inactivation (Fig. 1A). ID1 is made up by residues 1-37 (9), and ID2 resides between amino acids 40 and 68 (20). More explicitly, the existence of the second ID became evident after the first 39 residues were deleted, and the resulting mutant channels did not lack inactivation but rather displayed rapid inactivation very similar to that observed with ID1 present at the N terminus (20, 21). However, it remained obscure what the functional role of the second ID might be in intact channels and, therefore, how inactivation gating is realized by this tandem inactivation domain.

Here we analyze the solution structure and the function of this tandem inactivation domain of Kv1.4 channels (amino acids 1-75, Kv1.4N-(1-75)) using NMR spectroscopy together with patch clamp recordings in whole cell configuration and simulations of neuronal spiking behavior.

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Peptide Synthesis and Sample Preparation-- A polypeptide covering the 75 N-terminal amino acids of Kv1.4 was expressed as a histidine-tagged GST fusion protein in the Escherichia coli strain BL21(DE3) and labeled uniformly with 15N using 15N-ammonium chloride (98%) as a nitrogen source. The protein was purified to homogeneity via nickel-nitrilotriacetic acid-agarose (Qiagen) and glutathione-agarose chromatography. The GST fusion domain was chopped off with thrombin leaving three residues (Gly-Ser-Thr) at the N terminus of Kv1.4N-(1-75).

For NMR Kv1.4N-(1-75) protein was dissolved in a solution containing 50 mM sodium phosphate (90% H2O, 10% D2O (v/v), pH 4.4), 10 mM dithiothreitol, and 1 mM protease inhibitors. The final protein concentration of Kv1.4N-(1-75) was 1 mM. For 1H chemical shift referencing, 2,2-dimethyl-2-silapentane-5-sulfonate was added as an internal standard.

NMR Spectroscopy-- All of the NMR spectra of Kv1.4N-(1-75) were recorded at 283 K on Bruker Avance spectrometers. At 600 MHz proton frequency, the following spectra were recorded according to standard procedures: 1H-15N HSQC, 1H-15N total correlation spectroscopy-HSQC (spin-lock mixing time of 70 ms), 1H-15N NOESY-HSQC (mixing time 120 ms), HNHA, and a two-dimensional homonuclear NOESY (mixing time 150 ms) in D2O solution. Spectral windows of 9 and 24 ppm were employed in the 1H and 15N dimensions, respectively. 3JHNHalpha coupling constants were determined from the three-dimensional HNHA spectrum as described previously (22). The 15N relaxation measurements (T1, T2, 1H-15N NOE) were carried out at 500-MHz proton frequency as described previously (23) with overall delays between scans of 2 s for the T1 and T2 measurements and 3 s for the heteronuclear NOE experiment, respectively. The delays used for the T1 experiments were 10, 20, 40, 80, 160, 320, 640, 1280, and 2560 ms, whereas delays of 8, 16, 24, 40, 64, 96, 128, 160, 256, 320, 480, and 800 ms were used for the T2 experiments. The 15N relaxation data were analyzed according to the reduced spectral density mapping approach (24).

Hydrogen/deuterium exchange of the backbone amide groups of Kv1.4N-(1-75) was evaluated by 1H-15N HSQC spectra on a uniformly 15N-labeled sample after solvent exchange to 99.9% D2O using a NAP5 column (Amersham Biosciences). NMR data were processed with the XWIN NMR software (Bruker) and analyzed with the programs AURELIA (Bruker) and XEASY (ETH Zürich) (25).

Structure Calculation-- The solution structure of Kv1.4N-(1-75) was calculated with the DYANA program package (version 1.5) that uses a simulated annealing algorithm in the torsion angle space (26). Distance restraints were derived from a 15N-edited three-dimensional NOESY spectrum in H2O solution and a 1H-1H two-dimensional NOESY spectrum in D2O solution. Volumes of unambiguously assigned NOESY cross-peaks were converted into proton-proton upper distance limits by the caliba routine within DYANA. The dihedral angle Phi  of residues with a 3JHNHalpha coupling constant of <= 6 Hz was restrained to values between -90 and -30°. Structures from preliminary DYANA runs were used to recalibrate the distance restraints in an iterative manner. Stereospecific assignments were provided by the glomsa algorithm implemented in DYANA.

The final family of structures was generated in a calculation with 300 random starting structures and 12,000 annealing steps. The 25 best structures had values for the target function <0.55 Å2, no NOE violations >0.3 Å, and no violations of dihedral angle constraints. These structures were selected for further analysis and evaluation of structural quality with the programs DYANA and MOLMOL (27). The latter was also used to prepare the figures showing three-dimensional structures.

Electrophysiology and Molecular Biology-- Site-directed mutagenesis was performed with a standard cassette technique (28), and the mutations were verified by sequencing. For functional expression, Kv1.4 WT and mutant subunits were subcloned into pBK-CMV (29) and the cDNA (0.5 µg/µl) was injected into Chinese hamster ovary dhFr- cells with a Microinjector (Transjector 5246, Eppendorf, Germany) (30). For identification of successfully injected cells, 10 ng/µl pBK-CMV-GFP was coinjected as a reporter. Cells were grown in minimum essential medium-alpha (Invitrogen) supplemented with 10% fetal bovine serum (Biochrom, Berlin, Germany) and penicillin/streptomycin (100 units/ml, Invitrogen) at 37 °C and 5% CO2.

Cells showing GFP fluorescence were chosen for electrophysiological experiments 20-40 h after injection. Whole cell voltage-clamp recordings were performed with an Axopatch 200B amplifier (Axon Instruments, Foster City, CA) at room temperature (21-24 °C) using quartz pipettes with resistances of 1.5-2.5 megohms when filled with intracellular solution of the following composition (in mM): 135 KCl, 3.5 MgCl2, 0.1 CaCl2, 5 K2EGTA, 5 HEPES, 2.5 Na2ATP, and 0.1 dithiothreitol (pH adjusted to 7.3 with NaOH). Dithiothreitol was used to establish a stable redox milieu in the cytoplasm (9). After establishing whole cell configuration and series resistance compensation, residual series resistance was <0.8 megohms. Extracellular solution was (in mM) 144 NaCl, 5.8 KCl, 1.3 CaCl2, 0.9 MgCl2, 10 HEPES, 0.7 Na2HPO4, and 5.6 glucose (pH adjusted to 7.3 with KOH). For competition experiments, TEA-Cl (1 mM) was added to the intracellular solution and exchange of the intracellular solution was achieved by successively repatching the same cell with pipettes containing different intracellular solutions as described previously (30). The absence of channel run-down during successive experiments was verified by concluding the experiment with the control solution used initially.

Activation and inactivation kinetics of Kv1.4 channels (Figs. 4-6) were measured by stepping the membrane potential from -120 mV to the values indicated for variable periods (between 250 and 500 ms). Steady-state inactivation curves as in Fig. 6B were obtained with a standard two-step protocol, which used a prepulse of 0.8 s to potentials between -120 and 20 mV (10-mV increments) and a test pulse to 60 mV that determined the fraction of channels inactivated during the prepulse. Between individual cycles, the voltage was held at -120 mV to allow for complete recovery from inactivation. The inactivation curve was fitted with a Boltzmann function (I = [1 + exp{(V - V1/2)/k}]-1), where V is the membrane (prepulse) potential, V1/2 is the potential at half-maximal inactivation, and k is the slope factor. Activation curves (used for the simulations in Fig. 7) were obtained by stepping the membrane potential from -120 mV to potentials between -80 and 60 mV (10-mV increment) for 250 ms. The current amplitude was divided by the driving force to yield channel activation as a function of membrane potential.

The time course of recovery from inactivation was obtained with a three-step voltage protocol. 1) Voltage was first stepped to 60 mV for 1 s to allow for complete inactivation. 2) Voltage was then stepped to -120 or -80 mV for a variable period. 3) The final step used to determine the fraction of channels that recovered during the hyperpolarizing pulse was to -20 mV.

Currents were low pass filtered at 2 kHz (4-pole Bessel) and sampled at 10 kHz. Processing and fitting of the data were performed with IgorPro (Wavemetrics, Lake Oswego, OR). All of the data are given as the mean ± S.D. of n experiments.

Simulation of Neuronal Signaling-- Simulations of neuronal input-output relations were performed with Neuron (version 4.3.1) (31, 32). This simulation environment calculates the voltage response of a cell (of given geometry) to current stimuli based on Hodgkin-Huxley-type voltage-gated conductances for Na+ and K+ (non-inactivating K+ currents) and standard values for leakage conductance and passive electrical properties including membrane capacitance and intracellular resistance. To implement the kinetics of Kv1.4 WT and (Delta 40-50) mutant channels, the measured steady-state activation and inactivation curves and the recovery from inactivation as well as the time constants for activation and inactivation on a broad voltage range (-100 to 80 mV) were used to determine the Hodgkin-Huxley parameters of channel gating (33). The following rates were used to model the inactivation gating of Kv1.4 WT (alpha h(V) = 0.000025608·exp(-V/45.4217), beta h(V) = 0.0330402/exp(-(V + 45.6599)/2.30235 + 1)) and of Kv1.4-(Delta 40-50) (alpha h(V) = 0.000169537·exp(-V/223.943), beta h(V) = 0.0090891/exp(-(V + 43.7084)/2.63186 + 1). The voltage-gated Na+ conductance was 0.12 S/cm2, and the values for voltage-gated K+ and leakage K+ conductance were 0.036 and 0.0003 S/cm2, respectively. The membrane capacitance was 0.7 microfarad/cm2.

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Assignment and Backbone Dynamics of Kv1.4N-(1-75)-- A polypeptide containing the first 75 amino acids of Kv1.4 (Kv1.4N-(1-75)) was overexpressed in bacteria, purified, and used for structural analysis. A series of multidimensional (two- and three-dimensional) homonuclear and heteronuclear NMR experiments (see "Materials and Methods") was performed in aqueous solution under physiological salt conditions. The 1H and 15N resonances of the polypeptide were completely assigned by standard procedures based on 15N-edited three-dimensional total correlation spectroscopy and NOESY spectra.

As shown in Fig. 1B, the observed NOE connectivities displayed a distinct pattern along the Kv1.4N-(1-75) sequence with NOEs between non-adjacent amino acids (i,i+x) only occurring on the sequence stretch extending from Gly-20 to Ala-50. In this region, NOEs between the alpha -proton of one residue and the amide (dalpha N(i,i+3)) or beta -proton (dalpha beta (i,i+3)) of the third amino acid following were observed together with weak sequential contacts (dalpha N(i,i+1)) and strong amide-amide NOEs (dNN(i,i+1)), a NOE pattern usually seen with alpha -helices. In line with this interpretation, all of the 3JHNHalpha coupling constants determined for residues 20-50 were between 4 and 5.6 Hz (Fig. 1B), values regarded diagnostic for helical conformation (34), whereas the scalar couplings for the residues N- and C-terminal to this "core region" of Kv1.4N-(1-75) were between 6 and 8 Hz, as typically seen for random coil peptides (35).


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Fig. 1.   Functional domains of Kv1.4 and NOE contacts observed for Kv1.4N-(1-75) in aqueous solution. A, Cartoon illustrating trans-membrane topology and functional domains identified in Kv1.4 channels. ID1 and ID2 represent inactivation domains as identified earlier (9, 20), T1 (50) and NAB (51) are domains involved in subunit assembly and binding of Kvbeta subunits, respectively. B, NOE connectivities and J-coupling constants observed for Kv1.4N-(1-75) in aqueous solution. Sequential and medium-range NOEs are shown as a function of the amino acid sequence. Intensity of NOEs is represented by the line thickness. Filled circles represent 3JHNHalpha coupling constants between 3.9 and 6 Hz.

This view of structurally distinct parts in Kv1.4N-(1-75) was further supported by the chemical shift index of the alpha -protons as well as the backbone dynamics. Thus, most of the alpha -protons within the core region are shifted upfield by 0.1 ppm or more with respect to random coil chemical shifts (Fig. 2, upper panel). The backbone dynamics on the picosecond to nanosecond time scale as determined from 15N relaxation and the 1H-15N heteronuclear NOE (36) strongly suggested an ordered core region flanked by flexible N and C termini. As illustrated in Fig. 2 (middle and lower panels), the termini displayed very short transverse relaxation rates (R2) and negative values for the heteronuclear NOE, whereas residues 20-50 exhibited positive values of the 1H-15N NOE and relatively high R2 rates. Moreover, the spectral density functions J(0) and J(omega H) demonstrated reduced mobility of the protein backbone between residues 20 and 50 together with high mobility in the rest of the polypeptide (data not shown) (24).


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Fig. 2.   Dynamics of Kv1.4N-(1-75) reveal an ordered core flanked by flexible N and C termini. Upper panel, deviation of Halpha chemical shifts from random coil values. Delta delta Halpha was calculated as the difference between the random coil delta Halpha and the experimentally determined delta Halpha . Middle panel, ratio of transversal (R2) and longitudinal (R1) relaxation rates. Lower panel, 1H-15N heteronuclear NOEs (measured at 500 MHz). Increasing positive values indicate decreasing flexibility of the backbone (36).

It should be noted at this point that the NOE pattern, the Halpha chemical shifts, the scalar couplings, and the relaxation data suggest that the helical conformation may not extend throughout amino acids 20-50. Rather, there may be two helices with a different stability: the more stable one formed by amino acids Tyr-21 to Ala-38 (average deviation of 3JHNHalpha from random coil values of -1.7 ± 0.6 Hz); the second one involving the alanine-rich stretch between residues 40 and 50 (average deviation of 3JHNHalpha from random coil values of -0.8 ± 0.3 Hz).

Together, NOE pattern, scalar couplings, chemical shift data, and relaxation measurements suggest that Kv1.4N-(1-75) consists of a highly ordered core region with mostly helical conformation flanked by flexible N- and C-terminal ends. The absence of any stable tertiary folding is indicated by the small chemical shift dispersion of the backbone amide protons (~1 ppm), the fast H/D exchange of all amide protons in D2O solution, and the missing of long range NOEs in the NOESY spectra of Kv1.4N-(1-75).

Solution Structure of Kv1.4N-(1-75)-- A total of 391 experimentally determined distance restraints together with the restraints for dihedral angles and stereospecific assignments of protons (Table I) were used to calculate the solution structure of Kv1.4N-(1-75). After structure calculations with the simulated annealing protocol of DYANA (26) in the torsion angle space, a family of 25 structures with lowest values of the target function and without NOE violations >0.3 Å was selected as the final family of Kv1.4N-(1-75) structures (see Table I for structural statistics).


                              
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Table I
Structural statistics for the family of 25 Kv1.4N-(1-75) structures
All 75 residues are included in the analysis unless otherwise noted.

Fig. 3 shows 22 representatives of this family of best structures superimposed either between residues 21 and 37 (Fig. 3A) or residues 41 and 49 (Fig. 3B). Both superpositions converged well with similar root mean square deviation values to the mean structure (0.50 ± 0.30 Å and 0.76 ± 0.34 Å for the backbone atoms in Figs. 3A and B, respectively), whereas superposition of the entire core region was not meaningful because of the divergent orientation of Ala-38 and Ala-39 that link the two convergent stretches (see Table I). Thus, the double Ala linker divides Kv1.4N-(1-75) into an N- and C-terminal domain. The N-terminal domain, which coincides with ID1 (9), comprises a flexible N terminus formed by the first 20 amino acids and a 5-turn alpha -helix between residues 21 and 38 (Fig. 4A). This structure is similar to the 37 amino acid piece we reported earlier (18) with the exception that the alpha -helix is extended by 2 turns and bent in its center. The C-terminal domain of Kv1.4N-(1-75) equivalent to the stretch harboring ID2 (20) consists of a 2.5-turn alpha -helix between amino acids 41 and 49 connected to a flexible domain formed by residues 50-75 (Fig. 4A).


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Fig. 3.   Solution structure of Kv1.4N-(1-75). A, backbone traces (N, Calpha , and C' atoms) of the family of best structures of Kv1.4N-(1-75) (with lowest target function) between amino acids 15 and 53 superimposed between residues 21 and 37 (highlighted in red). B, backbone traces as in A, but superimposed between residues 41 and 49 (highlighted in green). Superpositions depict 22 of the 25 best structures of Kv1.4N-(1-75) (see "Solution Structure of Kv1.4N-(1-75)" under "Results"). N and C termini are indicated.


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Fig. 4.   Structure-function relation of fast inactivation in Kv1.4. A, N-terminal 53 residues of the best Kv1.4N-(1-75) structure in ribbon representation emphasizing the secondary structural elements between residues Gly-20 and Ala-38 and alanine residues 40 and 50. B, deletion analysis illustrating the functional significance of the structural domains shown in A. Traces are current responses to a voltage step from -80 to 20 mV recorded in Chinese hamster ovary cells injected with Kv1.4 WT or the deletion mutants indicated. Scaling is 100 ms and 1 nA for all of the traces. Note that fast inactivation is only observed with either residues 1-38 (ID1) or 39-50 present at the N terminus of the channel.

Structure-Function Analysis Delineates Distinct Properties and Contribution to Fast Inactivation of ID1 and ID2-- The structure presented above (Fig. 4) was used as a guide to delineate the functional properties of the tandem inactivation domain of Kv1.4 channels. For this purpose, the distinct structural domains were deleted and the respective mutants were expressed in Chinese hamster ovary cells and probed for their inactivation characteristics. Fig. 4B depicts representative current responses to a 250-ms voltage step from -80 to 20 mV. Similar to earlier reports, rapid and complete inactivation was observed with WT channels bearing ID1 at its N terminus as well as with the Delta 2-38 mutant in which the ID1 stretch was deleted (20). In either case, the inactivation time constant (tau inact) at 20 mV was ~16 ms (tau inact of 16.1 ± 2.6 ms (n = 6) and 15.8 ± 2.7 ms (n = 7) for WT and Delta 2-38, respectively). More detailed analysis showed that in WT, rapid inactivation was largely impaired by deleting either the flexible domain of ID1 formed by residues 1-20 or the 5-turn alpha -helix (Fig. 4B, left panel). In contrast, fast inactivation in the Delta 2-38 mutant predominantly required the alpha -helical domain between amino acids 40 and 50. Its deletion completely abolished inactivation, whereas further deletion had no additional effect (Fig. 4B, right panel).

In addition to being susceptible to N-terminal deletions, rapid inactivation in WT as well as in the Delta 2-38 mutant fulfilled another hallmark of the ball-and-chain mechanism: inactivation was competed by the open channel blocker TEA (16, 37, 38). As shown in Fig. 5A, 1 mM TEA slowed down the inactivation time course in either channel perfectly by the amount that would be predicted, assuming direct competition between TEA and the respective N-terminal ID for an overlapping receptor site within the open channel pore (39).


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Fig. 5.   Distinct inactivation properties of WT and Delta 2-38 mutant channels. A, inactivation of both WT and the Delta 2-38 deletion mutant is competed by 1 mM TEA applied to the cytoplasmic side of the channels. Left panel, current response to voltage steps from -80 to 20 mV recorded in the absence (gray) or presence (black) of TEA. Current and time scales are 1 nA and 50 ms, respectively. Right panel, relative increase in inactivation time constant (mean ± S.D.; n = 3) induced by 1 mM TEA. Line represents theoretical dependence of Delta tau inact on the fraction of channels blocked by TEA, assuming competition between TEA and the inactivation domain (Delta tau inact = 1/(1 - x), where x denotes the fraction of blocked channels (39)). B, representative recovery from inactivation recorded at a membrane potential of -120 mV, with a conditioning pulse of 1 s to 60 mV (for protocol see "Materials and Methods"). Line represents fit of a monoexponential to the recovery time course. Values for the time constant were 2.8 and 195.9 s for WT and Delta 2-38, respectively.

These results indicated that both ID1 and ID2 are able to inactivate Kv1.4 channels via occlusion of the open channel pore and thus raised the question of which domain may work as the actual ball domain. To answer this question, recovery from inactivation, i.e. unbinding of ID1 and ID2 from the receptor site, was investigated with a standard three-step protocol (see "Material and Methods"). As depicted in Fig. 5B, WT channels could be recovered completely by a hyperpolarizing pulse to -120 mV. The time course of recovery was well described by a monoexponential with a time constant of 2.75 ± 0.52 s (n = 6). In contrast, the unbinding of ID2 was roughly one hundred-fold slower than that of ID1, and recovery from inactivation could hardly be achieved in the Delta 2-38 mutant channels, even with hyperpolarizing pulses of >4.5-min duration (Fig. 5B; the respective time constant as extrapolated from such experiments was 295 ± 141 s (n = 3)). Such slow recovery, which by far exceeds the value obtained with any other Kv channel, reflects very slow dissociation of ID2 from its receptor site and may result from the particular "sticky" character of the polyalanine stretch in the ID2 alpha -helix.

Together, these results strongly suggest that ID1 rather than ID2 is the pore-occluding domain of Kv1.4 channels. But as so, what is the function of ID2 within the structural context of the Kv1.4 protein?

This question was assessed by site-directed mutagenesis that either deleted the ID2 alpha -helix or changed two consecutive residues of the ID2 alpha -helix to proline to induce a helix break at defined positions. The resulting mutants were then investigated for their gating properties. As illustrated by the current response in Fig. 6A, deletion of the polyalanine helix considerably slowed down channel inactivation, while the activation process remained unaltered. More detailed analysis showed that tau inact was increased by more than a factor of 4 on the voltage-range >= 0 mV where the inactivation time course is independent of channel activation and steady-state inactivation is complete (Fig. 6B). A similar approx 4-fold increase of tau inact was observed when proline residues were introduced at either the N-terminal end or the middle of the ID2 alpha -helix (A42/A43P and A45/A46P), whereas only a small increase was seen with prolines replacing amino acids 49 and 50 at the C-terminal end of the ID2 helix (Fig. 6C). Moreover, tau inact remained unchanged when the stretch between amino acids 50 and 60 (Delta 50-60) was deleted (Fig. 6C).


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Fig. 6.   Time course of inactivation is accelerated by the ID2 helix. A, current responses as in Fig. 5 for WT and Delta 40-50 mutant channels (scales as indicated). Inset, first 60 ms of the current response. Note the significant slowing in inactivation mediated by the deletion of the helical domain between residues 40 and 50. B, inactivation time constant (mean ± S.D. of seven experiments) as a function of membrane potential. Line is fit of equation A0 + A1 × exp(-bV) to the data (values for A0 and b were 15.8 ms and 9.6 mV for WT and 66.0 ms and 7.7 mV for Delta 40-50 mutant channels). Inset, steady-state inactivation of WT and Delta 40-50 mutant channels. Values are mean ± S.D. of five experiments. Line is fit of a Boltzmann function to the data with values for V1/2 and the slope factor of -58.3 and 4.2 mV for WT and -47.7 and 4.3 mV for Delta 40-50. C, relative change in the inactivation time constant observed with a proline-scan of the ID2 helix. Inactivation time constants obtained with the mutants indicated (6-8 experiments each) were divided by the mean value of the tau inact of WT channels. Relative changes are given as mean ± S.D. Note that deletion of amino acids 50-60 had no effect on tau inact. D, recovery from inactivation at a membrane potential of -80 mV. Line represents fit of a monoexponential to the recovery time course with values for the time constants of 6.5 and 5.3 s for WT and Delta 40-50, respectively. Note the close match in recovery for both channels.

Different from the Delta 2-38 mutant, recovery from inactivation in the Delta 40-50 channels was basically unchanged with respect to WT. Thus, a monoexponential fit to the recovery time course at -80 mV yielded mean values of 5.3 ± 0.7 s (n = 7) and 5.9 ± 0.5 s (n = 3) for Delta 40-50 and WT channels, respectively (Fig. 6D). Consequently, the 4-fold increase in tau inact did not result from a change in dissociation of ID1 from its receptor. Instead, the rate of association between ID and receptor must be decreased. In the "sequential-step" model of inactivation (13, 17, 40), such association actually implies two separate steps: first, the approach of the ID toward the channel entrance (preinactivation) and, second, the binding of the ID to its receptor (inactivation). In this context, the alanine-rich helix in ID2 may be expected to work as a "docking domain" that binds the N terminus to the surface of the channel vestibule and thus promotes rapid insertion of ID1 into its receptor, which finally occludes the pore and inactivates the channel (see also "Discussion").

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REFERENCES

Structure-Function Correlation and Model for Fast Inactivation of Kv1.4-- Here we show that the solution structure of Kv1.4N-(1-75), a polypeptide covering the tandem inactivation domain of Kv1.4 channels (20), consists of two domains connected by a short flexible linker. The N-terminal half termed ID1 comprises a flexible region anchored at a 5-turn alpha -helix, and the C-terminal half is a 2.5-turn alpha -helix (ID2) followed by a disordered C-terminal tail (Fig. 4A). This structural bipartitioning correlates with distinct functional properties determined for the two domains in the context of the whole Kv1.4 protein. ID1 occludes the ion pathway consistent with the function of a classical ball domain, whereas ID2 most probably serves as a docking domain that pins ID1 to the inner surface of the channel and promotes its rapid access to its receptor site in the open channel.

These conclusions are based on several observations. First, although both ID1 and ID2 block the open channel when present at the N terminus (competition with TEA, Fig. 5A), they largely differ in their recovery from inactivation. Thus, recovery was identical and fast in all of the channels bearing ID1 at their N terminus (Figs. 5B and 6D). In contrast, recovery from ID2-mediated inactivation as observed in the Delta 2-38 mutant, occurred on a time scale >100-fold slower than that observed with ID1 (Fig. 5B). Second, channel inactivation was largely reduced by deleting either of the two distinct structural domains in ID1 with ID2 left unchanged in either molecule (Fig. 4B). Third, the removal of the ID2 helix or perturbation of its structural integrity (proline mutants) slowed the onset of inactivation ~4-fold (Fig. 6, B and C) but did not change the recovery from inactivation with respect to WT channels (Fig. 6D). These observations are most consistent with ID1 working as the "inactivation particle" and ID2 speeding the access of ID1 to its receptor without acting itself as a pore-occluding domain. As an alternative that is not completely ruled out by our experiments, ID2 might act as the actual ball domain whose high affinity is impaired 100-fold by ID1.

Together, structural and functional data would be consistent with a sequential model for fast inactivation in Kv1.4 channels. The hydrophobic ID2 helix may attach ID1 to the cytoplasmic surface of the channel protein, recently called preinactivation step (17), and thus promotes rapid insertion of ID1 into its "receptor site" that becomes accessible after opening (activation) of the channel pore. In Kv1.4, this ball- or N-type inactivation may subsequently trigger a collapse of the selectivity filter, a phenomenon known as C-type inactivation (41, 42). Recovery from inactivation, the reverse process of channel occlusion, should be initiated by dissociation of ID1 from its receptor, which finally reopens the pore. At present, the interactions between ID1 and the pore walls must remain speculative as well as the folding adopted by ID1 inside the channel (see Supplemental Fig. 1). Moreover, the binding site of ID2 on the cytoplasmic face of the channel protein remains to be elucidated.

Although most probably not relevant for inactivation, pore-occlusion by the short ID2 helix provides some new twists to where and how an ID might interact with the surface of the inner channel pore. We probed the insertion of this compactly folded piece into the recently proposed "open pore structure" of MthK/KcsA (43) using the docking procedure of the program GRAMM (44, 45). As illustrated in Supplemental Fig. 1, the ID2 helix is well able to enter the pore in helical configuration and to proceed until right underneath the selectivity filter, very similar to what was suggested for TEA derivatives (17).

Potential Significance of ID2 for Neuronal Signaling and for Kv1.4-mediated Short Term Plasticity-- Because rapid N-type inactivation is a major determinant for efficiency and short term plasticity of synaptic transmission, we probed the significance of the docking domain by simulating the input-output relation of a neuron using Neuron, an environment for solving nerve equations (31, 32). For this purpose, a spherical cell was equipped with a leakage K+ conductance, a standard voltage-gated Na+ conductance, and a voltage-gated K+ conductance, which either reflected non-inactivating Kv1.4 WT or Kv1.4-(Delta 40-50) channels (for details see "Material and Methods").

Fig. 7A illustrates spike responses of such a neuron elicited by a depolarizing current stimulus. Thus, with non-inactivating Kv channels, the cell responded with regular AP of constant amplitude throughout the stimulation period. In contrast, in the cell equipped with rapidly inactivating Kv1.4 WT, the stimulus elicited APs that successively decreased in amplitude and finally disappeared because of cumulative inactivation of the voltage-gated K+ channels. This means that Kv1.4 channels inactivated during the depolarizing phase of the AP cannot recover from inactivation during the repolarization period that is short with respect to the slow recovery time course (Figs. 5B and 6D). As a consequence, the cell remained depolarized once all of the Kv1.4 channels were inactivated.


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Fig. 7.   Functional significance of the docking domain probed by simulations with a model neuron. A, differential spiking behavior of a neuron equipped with either non-inactivating Kv channels (upper panel), rapidly inactivating Kv1.4 channels (middle panel) or Kv1.4 channels lacking the docking domain (Delta 40-50). Simulations were performed with the Neuron simulation environment (Neuron 4.3.1) for a spherical neuron exhibiting voltage-dependent Na+ and K+ conductances, as well as a leakage K+ conductance. Note cessation of spiking because of cumulative inactivation of Kv1.4 channels that is absent in non-inactivating Kv channels or the Delta 40-50 mutant. B, number of APs triggered by stimulation with current of varying amplitude from simulations as in A; filled symbols refer to Kv1.4 WT (squares) and Kv1.4(Delta 40-50) (circles), open circles refer to non-inactivating Kv channels. Note the decrease in AP number observed for rapidly inactivating Kv1.4 channels at higher stimulus amplitudes. C, memory effect of rapidly inactivating Kv channels envisaged by double-pulse excitation is largely impaired by deletion of the docking domain. Note the difference in response to the second excitation observed between Kv1.4 WT and mutant channels. Single pulse excitation of a Kv1.4 containing neuron is shown for comparison.

The phenomenon of cumulative inactivation appeared to be abolished in the absence of the docking domain, and the cell equipped with Kv1.4-(Delta 40-50) displayed a spiking pattern very similar to that observed with non-inactivating Kv channels (Fig. 7A, lower panel). As shown in Fig. 7B, cumulative inactivation and, concomitantly, the effect of the docking domain were dependent on the stimulus amplitude. The larger the stimulus amplitude, the more prominent it appeared.

In central nervous system neurons, cumulative inactivation implements a "molecular memory" that operates on the time scale of seconds (2, 46, 47). This memory effect is shown in Fig. 7C where a large amplitude excitation is preceded by a low amplitude stimulus. The AP frequency that is determined by the set of conductances in the Neuron platform (31, 32) was slightly above 100 Hz, in close agreement with what is known regarding the typical spiking of granule cells in the hippocampus (48, 49).

During this low amplitude stimulus, cells equipped with Kv1.4 WT or Delta 40-50 channels spike regularly with no obvious difference. However, the following excitation revealed marked differences between both channels. Kv1.4 WT cells "remembering" the preceding pulse only fire very few APs and then remain depolarized throughout the rest of the stimulus, whereas Kv1.4-(Delta 40-50) cells responded with continuous spiking as if no preceding pulse occurred. Thus, the docking domain appears to be crucial for the molecular memory generated by rapidly inactivating Kv1.4 channels.

    ACKNOWLEDGEMENTS

We thank Otogene AG (Tübingen) for providing access to the NMR spectrometer. We are particularly indebted to Dr. P. Jonas for help with Neuron and the simulations of the neuronal input-output relations shown in Fig. 7.

    FOOTNOTES

* This work was supported in parts by grants of the Deutsche Forschungsgemeinschaft (Fa332/3-1) and the Interdisciplinary Center of Clinical Research (IZKF) Tübingen (Project IA4) (to B. F.).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.

The on-line version of this article (available at http://www.jbc.org) contains Supplemental Fig. 1.

|| To whom correspondence should be addressed. Tel.: 49-761-203-5175; Fax: 49-761-203-5191; E-mail: bernd.fakler@physiologie.uni-freiburg.de.

Published, JBC Papers in Press, February 16, 2003, DOI 10.1074/jbc.M210191200

    ABBREVIATIONS

The abbreviations used are: Kv, voltage-activated K+ channel; AP, action potential; ID, inactivation domain; TEA, tetraethylammonium; NOE, Nuclear Overhauser Enhancement; GFP, green fluorescent protein; CMV, cytomegalovirus; WT, wild type; HSQC, heteronuclear single quantum coherence; NOESY, NOE spectroscopy.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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