1Department of Biophysics and Cell Biology, Research Center for Molecular Medicine, and 2Cell Biophysics Research Group of the Hungarian Academy of Sciences, Department of Biophysics and Cell Biology, Medical and Health Science Center, University of Debrecen, H-4012 Debrecen, Hungary; 3Pacific Biomedical Research Center, Bekesy Laboratory of Neurobiology, University of Hawaii, Honolulu, Hawaii 96822; and 4Department of Internal Medicine, Yale University School of Medicine, New Haven, Connecticut 06520
Submitted 10 October 2003 ; accepted in final form 10 June 2004
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ABSTRACT |
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extracellular pH; potassium channel; histidine; barium; high ionic strength
Raising the extracellular K+ concentration generally slows the rate of slow inactivation, which is attributed to a "foot-in-the-door" mechanism (1, 26). At high extracellular K+ concentration the occupancy of a K+ binding site within the pore is increased, and as a result, the structural changes leading to the inactivated state are delayed. This binding site is believed to be the outermost binding site in the selectivity filter, which has been referred to as the "external lock-in site" (11, 18, 19, 30).
Mutations in the S5-S6 linker, particularly in the pore region, can alter the rate of slow inactivation. The amino acid at the position corresponding to 449 in the Shaker channel, which is located at the external mouth of the pore, is an especially strong determinant of the inactivation rate in various K+ channels. Mutations at position 449 in Shaker produced drastic changes in the kinetics of slow inactivation (26). Introduction of alanine or charged amino acids at this position caused an increase, whereas histidine or amino acids with large hydrophobic side chains caused a decrease, of the inactivation rate. Raising external K+ concentration slowed inactivation of the former, but not the latter, mutants.
Because the effects of changes in pHo on the function of voltage-gated K+ channels have an important physiological relevance, several studies have investigated these effects in various K+ channels. Most studies have shown that lowering pHo increases the rate of slow inactivation and that pH sensitivity is enhanced by the presence of a histidine in the S5-S6 linker region. In wild-type Shaker channels inactivation was accelerated by low pHo, and this effect was also seen in most T449X mutants, with the most dramatic change occurring in T449H (26, 34). In addition, an F425H mutation in the turret also modified the pH dependence of the inactivation kinetics (34). Acidification of the external medium increased the inactivation rate in rat (r)Kv1.5 channels bearing a histidine (His452) in the S5-S6 linker (the equivalent of Shaker Phe425). However, inactivation of rKv1.2, which lacks this histidine, did not show pHo dependence (36). Slow inactivation of ferret (f)Kv1.4 was also sped up by acidosis through the equivalent residue, His508, of that channel (21). In addition, the mutation K532Y (equivalent of Shaker Thr449 and Kv1.3 His399) decreased the rate of slow inactivation and made it pH insensitive. Kehl and coauthors (17) found that low pHo also blocked outward K+ currents in human (h)Kv1.5 channels and that this blocking effect was reduced by increased external K+ concentrations. These authors concluded that the increased inactivation rate could not explain the decrease in current amplitude.
In the present study, we examined the effects of pHo on the rate of slow inactivation in hKv1.3 channels. The Kv1.3 channel is the dominant K+ channel in human lymphocytes. This channel inactivates only with the slow, but not the N-type, mechanism, so changes in the rate of slow inactivation can be studied in isolation. Inactivation of Kv1.3 resembles that of Shaker P/C-type inactivation in that it is slowed by elevated external K+ concentration and TEA via the foot-in-the-door mechanism (8, 9). However, in Kv1.3 there is a histidine residue in the critical position of the pore region (His399, equivalent of Shaker 449), which suggests that this channel may be physiologically modulated by pHo.
Deutsch and Lee (7) found that acidification of the external solution shifted the activation threshold to more positive voltages in Kv1.3 and reduced the K+ current amplitude, presumably due to the screening of membrane surface charges by protons (7, 17, 37). They also found that inactivation in Kv1.3 was slowed by low pHo. Because of the weak voltage sensitivity of the inactivation rate, the authors concluded that the voltage shift produced by external protons was not sufficient to explain this effect. Another study (2) confirmed the slowing of inactivation by low pHo in rKv1.3 (RGK5) channels and found that the mutation of His401 (the analog of His399 in hKv1.3) to Tyr significantly altered the inactivation kinetics of the channel. This indicates the involvement of residue His401 in the inactivation process similarly to its analog, Thr449, in Shaker. Further proof for the critical role of this residue in slow inactivation was provided by extensive mutational analysis of mouse (m)Kv1.3 channels, which showed marked changes in the inactivation kinetics of the mutant channels (15, 31). However, none of these studies suggested a model or characterized the mechanism by which external protons affect slow inactivation.
In this study we tested the hypothesis that exit rate of cations from the external lock-in site is influenced by the nature of the amino acid residue at position 399 and that it is the positive charge of protonated histidines that hinders the emptying of this site, thereby delaying inactivation. To test this hypothesis, we introduced nontitratable neutral or positive amino acid mutations at position 399 of hKv1.3, used a high-ionic-strength solution to reduce the range of electrostatic interactions, and measured the exit rate of barium ions from the wild-type and mutant channels at different pHo values. On the basis of our results, we present a model in which a reduction of pHo causes the protonation of the crucial His399 residues, thereby creating an electrostatic energy barrier between the external bulk solution and the external lock-in site. The reduced rate at which potassium ions cross this barrier explains the slowing of inactivation in Kv1.3 at low pHo.
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MATERIALS AND METHODS |
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Cells.
Cytotoxic murine T cells (CTLL-2) were transiently cotransfected with plasmids for CD4 and for one of the hKv1.3 channels at a molar ratio of 1:5 or 1:8 (32 or 48 µg/ml total DNA) by electroporation (6). CTLL-2 cells were cultured in RPMI-1640 supplemented with 10% FBS (Hyclone, Logan, UT), 2 mM Na pyruvate, 10 mM HEPES, 4 mM L-glutamine, 50 µM 2-mercaptoethanol, and 100 Cetus units/ml IL-2. Before transfection, cells were cultured for 24 h in fresh medium and collected in the logarithmic phase of growth. After harvesting, cells were suspended in Hanks'-20 mM HEPES balanced salt solution (pH 7.23) at 2 x 107 cells/ml and the appropriate mixture of DNA was added to the cell suspension. This suspension was transferred to electroporation cuvettes (400 µl/cuvette, 4-mm electrode gap), kept on ice for 10 min, and then electroporated with a BTX electroporator (San Diego, CA) with settings previously determined to give 50% viability at 24 h after transfection (725 V/cm, 2,350 µF, 13
). The resultant time constants were 2425 ms. Cells were incubated for an additional 10 min on ice and transferred back to culture medium (
0.5 x 106 cells/ml) supplemented with 5 mM Na-butyrate (37°C, 5% CO2). Cells were used for electrophysiology between 8 and 16 h after the transfection.
Electrophysiology.
Whole cell measurements were carried out with Axopatch-200 and Axopatch-200A amplifiers connected to personal computers using Axon Instruments TL-1-125 and Digidata 1200 computer interfaces, respectively. For data acquisition and analysis the pCLAMP6 and pCLAMP8 software packages (Axon Instruments, Foster City, CA) were used. CD4-positive CTLL-2 cells were selected for current recording by incubation with mouse anti-human CD4 antibodies (0.5 µg/106 cells; AMAC, Westbrook, ME), followed by selective adhesion to petri dishes coated with goat anti-mouse IgG antibody (Biosource, Camarillo, CA), as previously described by Matteson and Deutsch (27) and Deutsch and Chen (6). Dishes were washed gently five times with 1 ml of standard extracellular bath medium (see below) before the patch-clamp experiments. Standard whole cell patch-clamp techniques were used as described previously (10). Pipettes were pulled from GC150F-15 borosilicate glass capillaries (Clark Biomedical Instruments) in five stages and fire-polished to give electrodes of 2- to 3-M resistance in the bath. The standard extracellular bath solution (S-ECS) contained (in mM) 145 NaCl, 5 KCl, 1 MgCl2, 2.5 CaCl2, 5.5 glucose, and 10 HEPES (pH 7.35, 305 mosmol/kgH2O). The pipette solution was (in mM) 140 KF, 11 K2EGTA, 1 CaCl2, 2 MgCl2, and 10 HEPES (pH 7.20,
295 mosmol/kgH2O). Cells with low series resistance (25 M
) were used, and series resistance compensation up to 85% was applied to minimize voltage errors and achieve good voltage clamp conditions. The uncompensated series resistance error was
510 mV. The reference electrode was connected to the recording chamber with an agar bridge to eliminate junction potential changes during perfusion.
Test substances.
When external TEA was tested, we added equimolar TEA-Cl to replace NaCl. The high-ionic-strength (HIS) extracellular solution contained (in mM) 10 NaCl, 5 KCl, 96.25 MgCl2, 2.5 CaCl2, 5.5 glucose, 10 HEPES (pH 7.35, 320 mosmol/kgH2O). In the 15 mM Ba2+ solution NaCl was replaced by equimolar BaCl2. Bath solutions having low pH (6.5, 5.5) were buffered with 10 mM MES instead of HEPES.
Bath perfusion with different test solutions was achieved with a gravity-flow perfusion setup with eight input lines and a PE-10 polyethylene tube output tip with a flanged aperture to reduce the turbulence of the flow. The solutions were applied in an alternating sequence of control and test solutions unless otherwise stated. Excess fluid was removed continuously from the bath.
Data analysis. Before analysis, whole cell current traces were corrected for ohmic leak and digitally filtered (3-point boxcar smoothing). Nonlinear least-squares fits were done with the Marquardt-Levenberg algorithm. Fits were evaluated visually as well as by the residuals and the sum of squared differences between the measured and calculated data points. Statistical comparisons were made with ANOVA supplemented with Bonferroni t-test for pairwise comparisons, Student's t-test, and when appropriate, paired t-test at P = 0.05. For all experiments, the SE of the mean is reported.
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RESULTS |
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Acidification-induced shift in the voltage dependence of steady-state activation is demonstrated in Fig. 2A for Kv1.3 channels expressed in a CTLL-2 cell. A Boltzmann function was fitted to the data points, and the midpoint (V1/2) and slope (s) characterizing voltage dependence of steady-state activation were determined at different pHo values. Lowering pHo induced a significant shift in V1/2 toward depolarized voltages (P < 0.001, ANOVA), but s was the same at all tested pHo values (P < 0.72, ANOVA; Table 1).
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Sensitivity of inactivation to low pHo is related to residue His399. The most likely candidate for conveying changes in pHo to changes in the rate of inactivation in Kv1.3 is the histidine residue at position 399 near the extracellular end of the pore (2, 26). The acidic dissociation constant (pKa) of the histidine side chain in solution is 6.0, so it is likely to change its protonation state in the examined pH range. We used TEA to assess the extent of the protonation of His399 in Kv1.3.
Kavanaugh and colleagues (16) reported that the affinity of TEA for rKv1.3 channels was greatly reduced when the histidine residue at position 401 (corresponding to His399 in hKv1.3) was protonated in low pHo. This effect disappeared in the H401Y mutant, indicating the specific role of the protonation of His401. Levy and Deutsch (20) obtained similar results for Kv1.3 in human peripheral blood lymphocytes. Our results confirmed these observations. In Kv1.3 channels the extracellular application of 10 mM TEA resulted in the reduction of the current amplitude to 47 ± 3% (n = 4) of the control value at pH 7.35. In contrast, the current amplitude at pH 6.5 was 94 ± 4% (n = 5) of the control value in the presence of 10 mM TEA, indicating a significant degree of protonation of the His399 residues.
We obtained further evidence for the key role of the protonation of His399 residues in the pH dependence of Kv1.3 inactivation by testing several H399 mutants. We replaced the titratable histidines by residues that maintain their neutral or positive charge when the pH is changed between 7.35 and 5.5, thereby isolating the effect of the charge of this residue on the rate of inactivation without the background of any possible nonspecific pH effects.
In particular, we investigated how the rate of slow inactivation is affected by extracellular acidification in H399L, H399V, H399Y, H399K, and H399R. All of these mutant channels were expressed in high numbers in transfected CTLL-2 cells and were functional. Before examining the effect of pH on inactivation we compared some basic biophysical properties of these mutants with those of the wild-type channel.
At pH 7.35 the voltage dependence of steady-state activation (G-V function) of the neutral mutants (L, Y, and V) was similar to that of the wild-type channel (Fig. 2). V1/2 and s values of the G-V functions are shown in Table 1. However, the G-V function of the charged mutants (R and K) was shallower and V1/2 was shifted to more positive potentials (Fig. 2B, Table 1). This indicates a possible interaction of the residue at position 399 with the activation gating mechanism of this channel.
On exposure to a low-pHo solution, a shift of the G-V functions toward positive potentials was observed for each mutant, similarly to the wild-type channel. In addition, the G-V curves of H399K and H399R became steeper (Table 1). Activation rates were not affected by the mutations (data not shown; P < 0.232); however, the characteristics of inactivation changed dramatically in each mutant. All the tested mutants displayed biphasic slow inactivation; the decaying parts of the current traces were well fit by the sum of two exponential terms (Fig. 3). Table 2 shows the mean time constants for each mutant along with the relative weight of the slow component at pH 7.35.
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Ionic interactions are involved in slowing of inactivation at low pHo. In many K+ channels the rate of slow inactivation is known to be controlled by the occupancy of a potassium ion binding site in the permeation pathway (1, 18, 26). A possible explanation for the slowing of inactivation in low pHo demonstrated above is that the protonation of the His399 residues hinders the exit of potassium ions from this binding site through an electrostatic interaction.
As a test of this hypothesis we used a high-ionic-strength (HIS) extracellular solution to decrease the effectiveness of long-range electrostatic interactions. If the positively charged side chains of the histidines are responsible for slowing the exit of potassium ions from the binding site, the effectiveness of this interaction should be reduced in a HIS solution and faster inactivation should be observed.
The results shown in Fig. 4 support this hypothesis. When the external solution was switched from S-ECS pH 7.35 to pH 5.5 inactivation was slowed (Fig. 4A), but when the cells were bathed in HIS solutions switching from pH 7.35 to pH 5.5 caused an acceleration of the inactivation rate (Fig. 4B). Figure 4C shows the comparison from another aspect. Although switching from a normal to a HIS extracellular solution at pH 7.35 caused a reduction of current amplitude (59.2 ± 4.5%; n = 5, P < 0.001) and resulted in a higher steady-state current, it did not affect the inactivation rate (paired comparison: 107.9 ± 4.9%, n = 5, P < 0.182; control: = 167 ± 20 ms; HIS:
=179 ± 20 ms). In contrast, switching to a HIS solution at pHo 5.5 resulted in the acceleration of inactivation (paired comparison: 62.1 ± 5.8%, n = 4, P < 0.007; control:
=198 ± 23 ms; HIS:
=122 ± 15 ms) as well as current reduction (71.6 ± 4.6%; n = 4, P < 0.009). We did not investigate further the change in the current amplitude in HIS solution.
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The application of HIS solution also shifted the G-V relationship of the channels (Fig. 2A). V1/2 and s for the WT channels at pHo 7.35 were 14.4 ± 0.7 and 13.4 ± 0.5 mV, respectively (n = 3). The implications of this shift on our results are reviewed in DISCUSSION.
Low pHo slows barium dissociation from Kv1.3. We performed an additional test of our hypothesis using barium ions. Barium ions are similar in size to potassium ions and are able to enter the pore of several types of K+ channels from the extracellular side, but because of their divalent nature they are bound more strongly inside the pore and prevent permeation (14, 30). Hurst and coworkers (14) found that Shaker channels have two sequential binding sites for Ba2+: a more external site, which equilibrates quickly with the extracellular solution and has a low affinity, and a deep site with higher affinity and slower association and dissociation rates. The more external site, which is likely to be identical to the one termed the external lock-in site by Neyton and Miller (30), is thought to be that whose occupancy controls the rate of inactivation (11, 18, 19). According to our hypothesis, the protonation of the histidines should function as a barrier that would impede the exit of barium ions.
Extracellular application of 15 mM Ba2+ reduced K+ currents through wild-type Kv1.3 channels to 18.47 ± 1.28% of the control (n = 10). After achieving steady-state block, we measured the washout kinetics of Ba2+ after a switch back to a barium-free extracellular solution at pHo values of 7.35 and 5.5. Figure 5, A and B, demonstrates that dissociation of Ba2+ was slower at lower pHo. Time constants of dissociation were 66.49 ± 1.17 s (n = 5) and 129.42 ± 8.28 s (n = 5) at pHo 7.35 and 5.5, respectively (Fig. 5, E and F).
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DISCUSSION |
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In this study, we aimed to investigate the role of His399 in regulating the inactivation kinetics of Kv1.3 at low pHo. To study the role of protonation of this residue, we made point mutants of Kv1.3 having permanently positively charged (K, R) or neutral (L, V, Y) amino acids at this position and compared the properties of these mutants to those of the wild-type channel. We examined the pH and ionic strength sensitivity of inactivation kinetics, along with the dissociation rate of barium ions from the pore.
Characterization of wild-type and H399 mutant channels: surface charge screening and coupling of activation and inactivation.
The low pHo-induced voltage shift due to the surface charge screening effect was demonstrated earlier for endogenously expressed Kv1.3 in human peripheral blood lymphocytes by Deutsch and Lee (7). They reported a 35-mV shift to more depolarized potentials in the activation threshold of Kv1.3 when pHo was lowered from pH 7.4 to pH 5.5. This change is similar to that reported in the present study for the midpoint of steady-state activation of wild-type Kv1.3 channels expressed in CTLL-2 cells (
30 mV, Fig. 2). Mutation of His399 to neutral residues did not change the G-V function in normal pHo, and it did not affect the voltage shift caused by lowering pHo. In contrast, mutation to positively charged residues (R and K) shifted the G-V function in the positive direction and resulted in shallower voltage dependence. The midpoint for these mutants is similar to that obtained for hKv1.5, which has an arginine in the corresponding position (17). This raises the possibility of an interaction between pore residues and the gating machinery as suggested by Molina and coworkers (28). Switching to pH 5.5 reduced or abolished the differences in the G-V functions of neutral and charged mutants.
In Kv1.3 the voltage shift caused by extracellular acidification can account for the slowing of activation kinetics and reduction of current amplitude at intermediate voltages but not for the slowing of inactivation kinetics. This is supported by several arguments. First, although inactivation is coupled to activation, the several hundred-fold difference in the rates of these transitions rules out the possibility that the decreased activation rate in low-pH solutions is responsible for the decreased inactivation rate. This is further supported by the fact that the activation time constants of mutant channels with different inactivation kinetics were identical. Second, the inactivation rate of Kv1.3 was shown to be voltage independent at voltages that rapidly activate all channels, and therefore at a test potential of 50 mV the voltage shift does not influence the inactivation rate (3, 7). Third, the shift of the G-V function of the wild-type channel recorded in HIS solution toward more positive potentials was even greater than that caused by pH 5.5 (Fig. 2). However, in each case in which we applied a HIS solution we observed either acceleration or no change in inactivation kinetics as opposed to the slowing experienced at low pHo. In conclusion, a more direct mechanism must be responsible for the modulation of inactivation kinetics.
Characterization of wild-type and H399 mutant channels: inactivation at different pHo values.
The amino acid at position 449 in Shaker was shown to have a strong influence on inactivation kinetics (26). We found, in accordance with others (2, 31), that replacement of His399 by other amino acids had marked effects on inactivation kinetics in Kv1.3 as well. Direct comparison of the inactivation kinetics of the wild-type and mutant channels (and the changes induced by pHo) was hampered by the fact that all mutant channels displayed biphasic inactivation as opposed to the inactivation of the wild-type channel, which could be well fitted with a single-exponential function (Figs. 1 and 3). The overall rate of inactivation of H399L was very similar to that of the wild type, so we used this neutral mutant for further experiments. H399R and H399K inactivated at a significantly slower rate, strengthening the idea that a positive charge at this position in Kv1.3 slows inactivation. This is in clear contrast to Shaker, which inactivated very quickly with positive residues at the corresponding position (26). Of the mutants having permanently positive charge at position 399 we chose to use H399K for further experiments because lysine has a shorter side chain than arginine and thus steric effects should be smaller with lysine. The other two neutral mutants, H399Y and H399V, inactivated very slowly like their Shaker counterparts. This sequence of overall inactivation rates perfectly matches the results obtained for equivalent mKv1.3 mutants (31). The facts that the T449H Shaker mutant reacted to pH changes differently from Kv1.3 and that the inactivation kinetics of positively charged mutants in Kv1.3 and Shaker were very different, whereas those of the tyrosine and valine mutants were similarly slow, indicate that the amino acid residue at position 399 (449 in Shaker) is not the sole determinant of inactivation kinetics. Several studies have identified other amino acid residues that can modulate P/C-type inactivation. For example, Phe425 of Shaker in the turret and the equivalent residue His463 in hKv1.5 were shown to be such residues (17, 34), as well as Ala413 in the S6 region of hKv1.3 and Asp386 in the pore helix of mKv1.3 (31, 33).
On exposure to pHo 5.5 the inactivation rate of only the wild-type channel became slower; in all mutants an overall increase was apparent. This was a consequence of the acceleration of the fast component of decay in all mutants but H399Y, for which the decrease of the time constant was not significant (Table 2). In addition, the amplitude of the slow component decreased in each mutant. We have not investigated the nature of this accelerating effect, but we attribute it to the same apparently consistent phenomenon that was described for several members of the Shaker family and for which the possible involvement of the aspartate residue at the selectivity filter was suggested as an explanation (35). Reduction of the current amplitude was consistently present in the wild-type and all mutant channels, except in H399Y, for which the reduction was negligible. Whether this reduction is related to the accelerated inactivation rate remains controversial. In Shaker, current reduction was attributed to the increased rate of inactivation (35), but a study on hKv1.5 concluded that accelerated inactivation cannot account for the decrease in current amplitude (17).
Model: electrostatic interaction between residue 399 and potassium ions in the pore.
The hypothesis that we tested in this study was that the positive charge that the histidines at position 399 gain when they are protonated in low pHo is responsible for the slowing of P/C-type inactivation of wild-type Kv1.3. Our model suggests that these positive charges raise the height of the potential barrier just outside the outermost K+ binding site via electrostatic repulsion and thus reduce the exit rate of potassium ions from the pore, which delays inactivation. We also assume that the generally observed acceleration of inactivation in low pHo described above is present in all mutants, as well as in the wild-type channel, but in the latter the electrostatic force of the His399 residues overrides this effect.
The slower intrinsic inactivation of the permanently positive mutants (H399K and H399R) compared with the unprotonated wild-type or H399L channels supports the model. Moreover, there is no discrepancy between the acceleration of the inactivation rate of H399K and H399R at low pHo and the model because the general accelerating effect in these mutants is not counteracted by the slowing effect seen in the wild-type channel as these residues do not change their protonation states when switching to pH 5.5.
The following sections discuss results demonstrated in this study that support the presented model. First, the presence of a positively charged amino acid at position 399 renders inactivation kinetics sensitive to the ionic strength of the solution. Second, the presence of a positively charged amino acid at position 399 slows Ba2+ dissociation kinetics from the pore. These results provide proof for the electrostatic nature of the effect and for the reduced exit rate of cations from the pore when hindered by a positively charged residue.
A positive charge at position 399 makes inactivation sensitive to ionic strength.
The electrostatic nature of the effect of the protonated histidines was studied with a HIS solution (Fig. 4). We showed that despite an increase in the steady-state current level inactivation kinetics of Kv1.3 channels was insensitive to the ionic strength of the extracellular solution at pH 7.35, where the protonation of the histidines is negligible. Inactivation of Kv1.3 is slower at pH 5.5 in normal extracellular solution, whereas acidification of the external medium in a HIS solution results in a significant acceleration of the inactivation rate. Thus shielding the electrostatic field of the protonated histidines unveils the normally observed accelerating effect of protons (possibly due to the titration of Asp397 in hKv1.3; see above).
We confirmed these observations by using mutants that had nontitratable residues in the place of the histidines. The application of HIS solution did not affect the inactivation rate of H399L, a neutral mutant, resembling the result obtained for the wild type at pH 7.35 (Fig. 4). In contrast, mimicking the situation of the wild type at pH 5.5, inactivation of H399K, a permanently positive mutant, was accelerated by HIS, presumably by reducing the lock-in effect of the positive charge on the potassium ion in the pore.
The HIS solution contained a high concentration of Mg2+, which might have other effects besides reducing the effectiveness of electrostatic interactions. Our measurements (Fig. 2A) confirmed the observation that increasing external Mg2+ concentration affects the voltage dependence of activation by surface charge screening just as the application of a low-pH solution does (37). However, as discussed above, this does not change inactivation kinetics as long as large depolarizations are used, as in our case, to +50 mV. This was verified by our measurements shown in Fig. 4C; inactivation kinetics were identical in S-ECS and HIS solutions at pH 7.35. Other divalent ions, such as Ca2+, could not be used for increasing ionic strength because they modulate the inactivation kinetics of Kv1.3 (8).
A positive charge at position 399 slows Ba2+ dissociation from the pore.
We used barium ions to monitor the occupancy of the external ion binding site, which is likely to be identical to the site whose occupancy by potassium ions controls the rate of inactivation (11, 18, 19). We showed that the dissociation rate of Ba2+ at low pHo is slower than at pH 7.35, because according to our model the protonation of the histidines impedes the exit of barium ions (Fig. 5). The results with the mutant channels supported our hypothesis: Ba2+ dissociation time constants were pH independent in the H399L mutant and were similar to those of the wild-type channel at pH 7.35, indicating a lack of a strong interaction between the exiting barium ion and residue 399. Conversely, time constants obtained with H399K were also pH independent but were similar to those measured for the wild-type channel at pH 5.5, implying that the repulsive interaction between the positive lysine residue and the barium ion hinders the exit of the latter from the pore.
We argue that a similar effect hindering the exit of potassium ions from this site can be responsible for the slowing of the inactivation kinetics at low pH. It is possible that the rate-limiting step in the slow dissociation of Ba2+ is not its exit from the external lock-in site but rather the transition from the deep site to the external lock-in site (14, 29, 30). However, even in this case, an indirect slowing effect of the protonated histidines (or lysines) is still possible through the hindrance of the dissociation of the potassium ion from the external lock-in site, the occupancy of which locks Ba2+ in at the deep site.
The explanation for the extremely long time constants of barium washout (on the order of minutes) is the following. Barium dissociation is very slow from the closed channels at negative holding potentials but becomes much faster from open channels at depolarized potentials (11). Because of the slow recovery of Kv1.3 channels from inactivation, depolarizing pulses were delivered every 30 s, which, combined with the 15 ms-long depolarizing pulses, results in short residency of the channels in the open state and consequent long washout time constants. The Ba2+ washout kinetics reported in this study is similar to those reported for Kv1.3 (8) and Shaker (11, 14) obtained with comparable voltage protocols.
In conclusion, the experimental results demonstrated in this study provide strong arguments for the importance of the amino acid residue at position 399 in controlling the exit rate of ions from the conduction pathway. As proposed by Kiss and Korn (18), it is conceivable that it is not directly the external lock-in site whose occupancy is modulated by the histidines but a more external K+ binding site of lower affinity. In this case, the more external site would serve as the lock-in site and, when occupied, it would lock the potassium ion in the selectivity filter at the control site and alter the inactivation rate. However, such modification of our model would not affect its other aspects and its consistency with the experimental results presented here.
The special feature of Kv1.3, that lowering pHo slows its inactivation kinetics, may be important in pathophysiological processes. At sites of inflammation, for example, pHo is considerably lower than the physiological pH in the extracellular fluid. If inactivation of Kv1.3 were faster with decreasing pHo, as for many Shaker-related K+ channels, the available K+ current for the regulation of the membrane potential of T cells would be diminished and the physiological response of the T lymphocytes would be impaired as well (22).
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GRANTS |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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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.
* S. Somodi and Z. Varga contributed equally to this work.
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REFERENCES |
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