Address correspondence to Karl L. Magleby, Department of Physiology and Biophysics, R-430 University of Miami School of Medicine, 1600 N.W. 10th Avenue, Miami, FL 33136. Fax: (305) 243-6898; email: kmagleby{at}miami.edu
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ABSTRACT |
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Key Words: Ca2+-activated K+ channels ring of charge conductance fast block proton block
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INTRODUCTION |
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Some of this material has appeared in abstract form (Brelidze et al., 2003a).
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MATERIALS AND METHODS |
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Xenopus laevis oocytes were separated by enzymatic treatment as described (Dahl, 1992; Hsiao et al., 2001
). The cRNA was transcribed using mMessage mMachine kit (Ambion) and injected in Xenopus laevis oocytes at
0.52 ng per oocyte, 28 d before recording. After injection, oocytes were kept at 1416°C in modified OR2 solution: (82.5 mM NaCl, 2.5 mM KCl, 1 mM CaCl2, 1 mM MgCl2, 1 mM Na2HPO4, 50 mg/l penicillin/streptomycin (Sigma-Aldrich), 50 mg/l gentamicin (Gibco), 4.8 mM HEPES, pH 7.5). The vitelline layer of injected oocytes was manually removed before patch clamp recording.
The mutant cDNA construct with the double mutation E321N and E324N was made using the Quickchange XL site-directed mutagenesis kit (Stratagene) and checked by sequencing (DNA Core Lab Sequencing Facility, University of Miami School of Medicine), as described previously (Brelidze et al., 2003b).
Solutions
Unless indicated, the extracellular (pipette) and intracellular solutions contained (mM): 150 KCl, 5 N-tris[Hydroxymethyl]- methyl-2-aminoethane-sulfonic acid (TES) to buffer pH (adjusted to pH 7.0), and 1 ethylene glycol-bis(2-aminoethyl)-N,N,N',N'-tetra-acetic acid (EGTA) and 1 N-(2-hydroxyethyl)ethylenediamine-N,N',N'-triacetic acid (HEDTA) to bind Ca2+ to prevent possible Ca2+ block from contaminating Ca2+ (Ferguson, 1991). The intracellular solution also typically contained 50 µM (+)-(18-crown-6)-2,3,11,12-tetra-carboxylic acid (crown ether) to bind Ba2+ to prevent Ba2+ block of the channel (Vergara and Latorre, 1983
; Diaz et al., 1996
). The extracellular (pipette) solution also typically contained 60 µM GdCl3 to block endogenous mechano-sensitive channels (Yang and Sachs, 1989
). The concentration of intracellular K+ ([K+]i) was varied as indicated by changing the concentration of KCl. Extracellular [K+] was kept constant at 150 mM in all experiments unless indicated. TES was used as a pH buffer. The optimal pH range for buffering by TES is from 6.8 to 8.2, but weaker buffering extends the range from
6.0 to 9.0. In the experiments where the intracellular pH (pHi) was changed from 5.0 to 9.0, the pHi was found to remain sufficiently stable to carry out the experiments. To further test for proper buffering of pH, in a few experiments where indicated 5 mM TES was increased to 10 mM, and 10 mM propionic acid, 10 mM 2-[N-Morpholino]ethanesulfonic acid (MES), and 10 mM N-tris[Hydroxymethyl]methyl-4-aminobutane-sulfonic acid (TABS) were also added. The pH of the solutions was adjusted by adding KOH and HCl to avoid adding Na+. In a few experiments where indicated 5 mM 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA), which is less sensitive to pH than EGTA and HEDTA (Tsien, 1980
), was added to act as a Ca2+ chelator. At pH 5.0, 5 mM BAPTA plus the 1 mM EGTA and 1 mM HEDTA would reduce the expected contaminant Ca2+ of 510 µM in the solution to a free Ca2+ of 0.020.04 µM (calculated with MAXC, www.stanford.edu/~cpatton/maxc.html; Bers et al., 1994
). EGTA and HEDTA were from Fluka, propionic acid, KCl, HCl, and KOH from Fisher; TES, MES, TABS, GdCl3, and crown ether from Sigma-Aldrich; and BAPTA from Molecular Probes. Solutions were changed using valve-controlled, gravity-fed perfusion of a microchamber (Barrett et al., 1982
).
Single-channel Recording and Data Analysis
Single-channel currents were recorded from BK channels expressed in oocytes using the inside-out configuration of the patch-clamp technique (Hamill et al., 1981). Patch pipettes were prepared from borosilicate glass tubing (Warner Instruments). Pipettes were heat polished to obtain resistances of 1020 M
when filled with 150 mM KCl solution. Data were acquired with an Axopatch 200A amplifier, sampled every 3 µs using a Digidata 1200A and pClamp7 software (Axon Instruments, Inc.), and stored and analyzed on a Pentium III computer.
Membrane potentials from -200 to +200 mV were generated by computer through the application of voltages to the external command input on the Axopatch 200A. Voltages less than -200 mV and greater than +200 mV were obtained by application of the computer applied voltages together with a steady-state holding potential preset on the front panel of the Axopatch 200A. For instance to achieve +250 mV, +50 mV was applied through the holding potential on the Axopatch 200A and then a brief (40100 ms) +200-mV pulse was applied through the computer-controlled external command. For voltages from-50 to +50 mV gap-free acquisition mode was used. For voltages less than -50 mV and greater than +50 mV, the episodic acquisition mode of pClamp 7 was used. The recording system was tested with a model cell and found to give linear i/V curves from -400 to +400 mV.
Unitary currents were measured from all-point histograms of the current records. Fig. 1 A shows a representative single-channel current record as it appears on the screen of the program interface. The corresponding voltage jump is indicated below the record. Fig. 1 B shows the same record as in Fig. 1 A, but with the data at the ends of the recording removed for the histogram analysis. The peaks of the current histograms correspond to the closed (C) and open current levels when one (O1), two (O2), and three (O3) BK channels were open at the same time. The distance between dashed lines placed at the peaks of the all-point histograms divided by the number of current steps between the dashed lines gave the average unitary current of open BK channels present in the patch. Currents recorded from -50 to +50 mV were typically measured by manually setting cursors on the closed and open current amplitudes because the histograms were less likely to show clear peaks due to the low activity. Unitary current amplitudes obtained by these methods were subsequently averaged for estimates obtained from three or more different patches studied under the same conditions, with the error bars in figures indicating the SEM. The absence of visible error bars indicates that the SEM was less than the size of the symbol.
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The effective filtering ranged from 4 to 33 kHz, and had no effect on the measured unitary conductance over this range because the open and closed current levels were well defined. Experiments were performed at room temperature (2123°C), unless otherwise indicated.
Fitting the Experimental Data
To analyze the titration curves for the inhibition of unitary current by protons, the data were fitted with Eq. 1
![]() | (1) |
To examine whether incorporating competitive inhibition of H+ on the action of K+ into the Woodhull equation would account for the i/V curves at different pHi, we incorporated the voltage dependence from the Woodhull equation (Eq. 5 in RESULTS) into equations from enzyme kinetics, as described below. The unitary current i0 in the presence of substrate K+ can be described by the Hill equation
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The ratio of current in the presence (ib) and absence (i0) of competitive blocker is then given by the ratio of Eq. 2 to Eq. 3
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To account for voltage-dependent concentration of the ions within the electric field of the membrane (Woodhull, 1973), [H+] and [K+] in Eq. 4 are given by
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The fitting of equations to the experimental data was performed in Origin (Microcal Software, Inc.) using the standard least squares fitting method.
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RESULTS |
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Contaminating Ba2+ in the Solution Does Not Affect the Unitary Current Amplitude
Studies recording currents from patches of membrane containing large numbers of BK channels showed that contaminating Ba2+ in the intracellular solution can significantly reduce macro currents recorded from BK channels (Diaz et al., 1996). Single-channel studies have shown that the reduced currents result from a dual effect of Ba2+ on currents through BK channels: Ba2+ decreases the effective open probability of the channel by producing long lasting blocks (seconds) of the channel (Vergara and Latorre, 1983
; Miller et al., 1987
; Bello and Magleby, 1998
), and Ba2+ can decrease the unitary current by a fast block (Vergara and Latorre, 1983
; Bello and Magleby, 1998
). To test if contaminating Ba2+ is producing the sublinearity of currents that we observed at high voltage, we examined unitary currents with and without the Ba2+ chelator crown ether added to the intracellular solution. From Fig. 2 B (filled squares for no Ba2+ chelator and filled circles with Ba2+ chelator) it can be seen that removing the contaminating Ba2+ with the chelator had no effect on the unitary current. Thus, contaminating Ba2+ in the solution does not contribute to the sublinearity of unitary currents.
As a further test to explore whether contaminating Ba2+ might be reducing the unitary currents, we added 100 µM Ba2+ to the intracellular solution containing 1 mM EGTA and 1 mM HEDTA in the absence of crown ether. For these conditions Ba2+ produced occasional long blocks of the channel that became more pronounced with voltage, reducing the open probability, as expected (Vergara and Latorre, 1983; Miller et al., 1987
; Bello and Magleby, 1998
), but there was little change in the peak unitary currents (Fig. 2 B, open diamonds). For voltages >200 mV with this large amount of added Ba2+, a slow flickery block developed that reduced the average current, but not the peak unitary currents. Considering that the total contaminant Ba2+ is typically <0.1 µM (Diaz et al., 1996
; Bello and Magleby, 1998
), a thousand times less than the 100 µM Ba2+ added in this experiment, contaminant Ba2+ would not be expected to alter measurements of unitary current amplitudes. Nevertheless, 50 µM crown ether was added to the intracellular recording solutions to assure that Ba2+ was not a factor.
MeSO3- and HEPES Do Not Reduce Unitary Current Amplitudes
Although we did not use MeSO3- and HEPES in our experiments, with these ions in the intra- and extracellular solutions Cox et al. (1997) observed that the unitary currents through BK channels reached a maximum value of 32 pA at +120 mV with no further increase with increasing depolarization. To explore why Cox et al. (1997)
saw such pronounced saturation compared with our observations (Fig. 2), we examined the possibility that some ion present in their solutions that was not in our solutions may have blocked the channel in a voltage-dependent manner. For example, Guo and Lu (2000)
found that HEPES, a commonly used pH buffer, or some accompanying impurities could cause rectification in IRK1 K+ channels. We recorded single-channel currents using intracellular and extracellular solutions prepared as described by Cox et al. (1997)
, with the intracellular solution containing (mM): 140 KMeSO3, 20 HEPES, 2 KCl, 1 EGTA, and 0.84 µM free Ca2+, pH 7.2; and the pipette (extracellular) solution containing (mM): 140 KMeSO3, 20 HEPES, 2 KCl, 2 MgCl2, pH 7.2. Unitary currents recorded with these solutions (Fig. 2 B, open squares) were essentially unchanged from those observed in our solutions (Fig. 2 B, filled circles). Thus, some other factor, perhaps an impurity in the stock chemicals, is responsible for the difference in results between our observations and those of Cox et al. (1997)
. Consistent with our observations, Talukder and Aldrich (2000)
observed unitary currents of
58 pA at +260 mV, similar to the value we observed at this potential.
Intracellular Protons Decrease Unitary Currents of BK Channels in a Concentration-dependent Manner
Protons block ion channels from both the extracellular (Woodhull, 1973; Prod'hom et al., 1987
; Tytgat et al., 1990
; Daumas and Andersen, 1993
; Klockner and Isenberg, 1994
; Coulter et al., 1995
; Chen et al., 1996
; Benitah et al., 1997
; Morrill and MacKinnon, 1999
; Lopes et al., 2000
; Geiger et al., 2002
) and intracellular (Davies et al., 1992
; Daumas and Andersen, 1993
; Habartova et al., 1994
; Xu et al., 2000
; Nimigean et al., 2003
) surfaces. To investigate whether block by intracellular protons contributes to the sublinearity of the i/V curves in Fig. 2, single-channel currents were recorded over a range of intracellular pH (pHi). Fig. 3 A shows representative single-channel records at +250 mV for pHi ranging from 5.0 to 9.0. The unitary currents at pHi of 7.0 and 5.0 were decreased by
15% and
55%, respectively, from that at pHi 9.0. The unitary currents at pHi 8.0 and 9.0 were essentially the same, indicating that H+i at 10-8 M has little effect on the unitary current. Plots of unitary currents recorded at +250 mV versus pHi, as well as for currents recorded at +200 and +100 mV, are shown in Fig. 3 B. At pHi 4.0 the channel still functioned, but clear open levels were difficult to define due to the high levels of block. Thus, currents at pHi < 5.0 were not studied. Habartova et al. (1994)
have previously examined the effect of pHi from 5.5 to 8.0 on unitary current amplitudes for voltages up to +40 mV, with observations similar to ours over this range.
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The data in Fig. 3, A and B, were obtained with a single pH buffer (TES, pKa of 7.4). This was done to prevent the possibility of channel block from additional buffers, but has the disadvantage of weak buffering at the extremes of the pH examined. To test whether the single buffer controlled the pH adequately for our experiments, unitary currents were recorded in the presence of four buffers (10 mM each) to span the pH range: propionic acid (pKa 4.9), MES (pKa 6.1), TES (pKa 7.4), and TABS (pKa 8.9) and compared with data with 5 mM TES alone.
Representative single-channel records at pHi 5.0, 7.0, and 9.0 recorded at +150 mV are shown in Fig. 3 C, where increasing the concentration of protons decreases unitary current amplitudes with the four pH buffers, as observed with one buffer. Comparison in Fig. 3 B of data obtained with the four pH buffers (filled diamonds) to that obtained with one pH buffer (open squares), and comparison of Fig. 4 C to Fig. 4 A, showed a similar proton block over a range of voltages, indicating that the pH of the solutions was adequately buffered with the single buffer. Although there was no difference in the unitary current amplitudes when using one pH buffer or four pH buffers for voltages 150 mV, an apparent discrete block developed with the four buffer solution that became so prominent with depolarizations >150 mV that the openings became too brief to measure (unpublished data). Hence, Fig. 4 C presents data for
150 mV. The discrete block in the four buffer solution is not proton block, as it was observed at pH 9.0 as well. Because of the apparent discrete block with the four buffer solutions at large depolarizations, one pH buffer was used for all subsequent experiments except for a few experiments in the next section.
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Proton Block Is Voltage Dependent
If protons reduce unitary currents by acting inside the conduction pathway of the channel, then the reduction of currents by protons should be voltage dependent. To explore this possibility, unitary currents were obtained at pHi 5.09.0 and plotted against voltage in Fig. 4 A. As indicated previously, the superposition of the unitary currents at pHi 8.0 and 9.0 shows that concentrations of protons 10-8 M have little effect on the unitary conductance. The reduction of the unitary currents for the increasing concentrations of protons from pHi 7.0 to 5.0 is readily apparent. To examine to what extent the proton block is voltage dependent, the ratios of the unitary currents at pHi 7.0, 6.0, and 5.0 to the unitary currents at pHi 9.0, where there is no block, are plotted in Fig. 4 B. For each pHi the ratio decreases with voltage, suggesting that the site of action of protons could be within the electrical field of the membrane. While protons could reduce unitary current amplitudes by
60% at pHi 5.0 and +300 mV, the effect of protons was negligible at pHi 7.0 for voltages
100 mV. The average chord conductance of BK channels at +100 mV and pHi 9.0 (in the essential absence of proton block) was 340 pS (straight line, Fig. 4 A), the average cord conductance at +100 mV and pHi 7.0 was 326 pS (straight line, Fig. 2 B). A paired t test of the cord conductances at pHi 9.0 and 5.0 at +30, +50, and +100 mV indicated that this small difference in cord conductance was not significant (P > 0.05).
The Ca2+ buffering ability of the EGTA and HEDTA depends on pH (Miller and Smith, 1984). Even though we used solutions with zero added Ca2+, a small amount of contaminating Ca2+ would be expected in the recording solutions due in large part to the Ca2+ in the KCl salt, so that some Ca2+ would be displaced from the Ca2+ buffers at the lower pHis. To test whether this displaced Ca2+ was blocking the channel, some experiments were also performed with 5 mM BAPTA added to the solutions to chelate Ca2+ (see MATERIALS AND METHODS). The reduction of currents by protons was the same in the absence and presence of BAPTA (unpublished data). Thus, the greater decrease in unitary currents at lower pHi was not due to block by contaminating Ca2+.
Proton Block Contributes to the Sublinearity of Unitary Currents at High Voltages
To determine to what extent proton block contributed to the sublinearity of the i/V plots obtained at pH 7.0 (Fig. 2), we compared i/V plots obtained at pHi 9.0, where proton block was negligible, to the i/V plots at pH 7.0. From Fig. 4 A it can be seen that only 40% of the sublinearity with increasing voltage at pHi 7.0 arises from proton block. The remaining sublinearity, still observed at pHi 9.0 in the absence of proton block, suggests that factors in addition to proton block contribute to the sublinearity.
The Woodhull Model can Approximate the Proton Block at a Fixed pHi, but Cannot Describe the Proton Block Over a Range of pHi
Fig. 4 shows that protons block BK channels in a concentration- and voltage-dependent manner. To determine if this block is consistent with the assumption that protons bind at a site within the electric field to block the channel, we examined whether the Woodhull model (Woodhull, 1973) could describe the data. This model is given by:
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Proton Block of BK Channels Is Consistent with Competitive Inhibition of K+ by H+
To investigate if the proton block of BK channels is consistent with competitive inhibition of K+i by H+i, double reciprocal plots of unitary current versus K+i were analyzed, similar to studies of enzyme kinetics (Stryer, 1981). Fig. 7 A shows theoretical double reciprocal plots with no blocker, and with blocker present for competitive and noncompetitive inhibition. For noncompetitive inhibition the plots with and without blocker intersect on the x-axis, and for competitive inhibition they intersect on the y-axes at an x value of zero, where [K+]i is infinite (1/[K+]i = 0), because the infinitely high [K+]i fully relieves the block by the competitive inhibitor.
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To determine whether incorporating the voltage-dependent blocking mechanism from the Woodhull equation into a competitive inhibition model would account for the i/V curves at different pHi, we simultaneously fitted the data in Fig. 5 A obtained at three different pHs with Eq. 4 (see MATERIALS AND METHODS). The dashed lines in Fig. 5 C indicate that Eq. 4 does not describe proton block with n = 1.0 (Hill coefficient), and the continuous lines indicate that Eq. 4 provides a better description of the data when n = 0.57, although there are still differences between the observed and predicted i/V curves at the higher voltages and pHis. Thus, the simple competitive inhibition model represented by Eq. 4 does not adequately describe proton block. For this test of competitive inhibition, the fraction d of the electrical distance from the inside for the site of action of K+ and H+ was constrained to be the same, as would be the case if they acted at the same site. The value of d for the plots in Fig. 5 C was 0.2, but values of d between 0.2 and 0.5 produced essentially the same fits because of compensating changes in the kd for protons.
When d was allowed to differ for H+ and K+, which could be the case for allosteric action of proton block, then the data were still not described by Eq. 4 when n = 1.0 (Fig. 5 D, dashed lines), but allowing n to be <1.0 gave an excellent description of the data with: n = 0.46, d = 0 for K+, and d = 0.2 for H+ (continuous lines, Fig. 5 D). Equally good fits could be obtained over a range of d for K+ as long as there was a well defined distance between the d for H+ and the d for K+, given by dH = 0.2 + 0.84dK. For example, a fit visually indistinguishable from the continuous lines in Fig. 5 D was obtained with n = 0.46, d = 0.2 for K+, and d = 0.36 for H+.
Eq. 4 could also describe the effect of high K+ in relieving the proton block (dotted lines in Fig. 6 A). For this fit n = 1.6, the d for K+ = 0.2, and the d for H+ = 0.21. Once again, similar fits could be obtained over a range of d's. (The implications of the description of the data by Eq. 4 will be considered in the DISCUSSION.)
The competitive interaction between K+ and H+ and the high concentration of K+ (150 mM) compared with H+ (10-7 M) would contribute to the lack of significant proton block under physiological conditions. Projecting the line in Fig. 6 B suggests that proton block could be highly significant for lower concentrations of permeant ion. Consistent with this possibility for a different channel, competitive extracellular proton block of L-type Ca2+ channels for physiological concentrations of the permeant Ca2+ (2 mM) may be sufficiently pronounced to have physiological implications (Chen et al., 1996).
Is the Competitive Inhibition of K+i by H+i at the Ring of Charge?
BK channels have a ring of eight negative charges at the entrance to the intracellular vestibule that doubles the unitary conductance for outward currents (Brelidze et al., 2003b; Nimigean et al., 2003
). H+ might reduce (block) unitary currents by protonating or screening the negative charges forming the ring of charge so that less K+ is concentrated in the vestibule. With less K+ in the vestibule, less K+ would be available to transit the channel.
To examine to what extent proton block requires the ring of charge, we neutralized the negative charge in the ring of charge with the double mutation E321N/E324N, and compared proton block for mutated and wild-type (WT) channels. Fig. 8 B plots unitary current amplitudes for mutated channels recorded at pHi 9.0, 7.0, and 5.0 against voltage, and Fig. 8 A replots data from Fig. 4 A from WT channels. Two observations are immediately apparent. The unitary currents of the mutant channels for all pHis are reduced when compared with WT channels, and protons still block the mutant channel. The reduction in currents would be expected from previous findings that the ring of charge doubles the unitary currents through BK channels by concentrating K+ in the intracellular vestibule by an electrostatic mechanism (Brelidze et al., 2003b). The further reduction of the unitary currents in the mutant channel at pHi 7.0 and 5.0, when compared with pHi 9, indicates that protons still block in the absence of the ring of charge.
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The data for the mutant channel in Fig. 8 B were replotted in Fig. 8 D to obtain the pH titration curves. The titration curves obtained at the three different voltages were then simultaneously fitted with Eq. 1, giving a ki of 12.6 µM (apparent pKa of 4.9) and n of 0.29. These values can be compared with the apparent pKa of 5.1 and n of 0.48 for WT channels (Fig. 3 B).
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DISCUSSION |
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Proton Block of BK Channels Is Voltage Dependent
Protons can decrease the unitary conductance of ion channels by binding to one or multiple sites within the pore of the channels (Daumas and Andersen, 1993; Chen et al., 1996
; Benitah et al., 1997
; Morrill and MacKinnon, 1999
). A characteristic of the block of BK channels by intracellular protons is that the block is voltage dependent, increasing with depolarization. Woodhull (1973)
developed a model to describe voltage-dependent proton block of Na+ currents by H+ by assuming that the site of block was within the electric field of the membrane so that voltage concentrated H+ at the blocking site. The Woodhull model could simultaneously describe proton block of BK channels over a range of pHis for voltages <100 mV, but could not describe the block at higher voltages. The inability of the Woodhull model to describe the data for BK channels would be expected since we found a competitive inhibition of H+ on unitary K+ current amplitudes (Fig. 7), whereas the Woodhull model assumes that the blocking and conducting ions do not interact.
By incorporating the mechanism of voltage-dependent concentration of ions from the Woodhull model into a model for competitive inhibition that also allowed for cooperativity (Eq. 4), we were able to simultaneously describe the voltage and pH dependence of proton block for pHi from 5.0 to 9.0 and for voltages up to +300 mV with a single set of parameters (Fig. 5 D). For 150 mM [K+]i, the best fits were obtained when the electrical distance through the membrane field measured from the inside of the membrane was 20% greater for H+ than for K+, suggesting the site of proton block may be different from the site of action for K+. However, although Eq. 4 could also account for data obtained at two different [K+]i as well (Fig. 6 A), it did not adequately describe the experimental observations when simultaneously fitting data for 60 mM, 150 mM, and 3.4 M [K+]i over multiple pHis and voltages (unpublished data). Thus, although Eq. 4 can be used to provide a quantitative description of competitive proton block of BK channels over a range of voltage and pHi for a narrow range of K+i, this equation is best viewed as a semiempirical equation because it does not fully account for the effect of [K+]i because it does not take into account such factors as changes in the electric field introduced by the blocking and conducting ions themselves and the effect of the ring of charge on concentrating K+.
Since the Woodhull model and Eq. 4 could approximate the voltage dependence of the proton block, it is of interest to relate the electrical distance for the block determined with these equations to the known structures of K+ channels. The fractional electrical distance of proton block from the inside of the membrane for the Woodhull model ranged from 0.08 to 0.15, and for Eq. 4 the electrical distance for the site of proton action was 0.2 greater than for the site of action of K+. These numbers would be consistent with the blocking site for protons being located within the conduction pathway of the channel,
1020% of the electrical distance through the electric field of the membrane. Whether this places the blocking site deep in the vestibule at the entrance to the selectivity filter or within the selectivity filter itself is not clear. Electrostatic calculations indicate that there is little voltage drop in the intracellular vestibule of the bacterial MthK channels (Jiang et al., 2002b
). If this is also the case for BK channels, then the site of proton block may be just within the selectivity filter.
However, estimates of the electrical distance for intracellular block of BK channels by TEA place the fractional blocking site for TEA 0.26 from the intracellular side (Blatz and Magleby, 1984). This electrical distance for block by TEA is greater than for block by protons, even though the large size of TEA would be expected to exclude TEA from the selectivity filter (Hille, 2001
; Zhou et al., 2001
). Thus, it is difficult to relate electrical distance to physical structure, especially since the size and number of the ions themselves could alter the apparent electrical distance. If the proposed intracellular RCK domains of the BK channel (Jiang et al., 2002a
) extend the intracellular conduction pathway for BK channels, as proposed for the intracellular NH2 and COOH terminus for GIRK1 and KirBac1.1 channels (Nishida and MacKinnon, 2002
; Kuo et al., 2003
), then the extended conduction pathway might also contribute to the electrical distance.
Our observation that proton block of BK channels is a fast block with no detectable increase in the open-channel noise does not rule out direct channel block. As pointed by Woodhull (1973), proton binding to proteins can be so fast (Fersht, 1985
) that single binding (blocking) events would not necessarily be detected for the frequency response of our experiments. Thus, direct channel block could occur without detectable increases in open-channel noise.
An observation of voltage-dependent block, however, does not require that the block occurs within the electric field of the membrane. Voltage-dependent block could also arise from the coupling of a blocker outside of the electric field to the movement of permeant ions inside the electric field. Such coupling is consistent with the voltage-dependent block of K+ channels by extracellular TEA and Ca2+i (Armstrong, 1971; Gomez-Lagunas et al., 2003
; Thompson and Begenisich, 2003
). The coupling works as follows. When the average position of K+ in the selectivity filter is near the extracellular side of the channel, the positive charge of K+ electrostatically repels the blocker from the extracellular blocking site. Shifting the membrane potential more negative then moves the average position of K+ toward the inner side of the selectivity filter away from the blocking site so that the blocker can now bind. Since the extracellular vestibule is so shallow (Jiang et al., 2002a
,b
), the extracellular blocker can be close to the entrance of the selectivity filter while remaining outside of the electric field. Thus, the voltage-dependent movement of K+ within the selectivity filter gives voltage dependence to a blocker outside of the electric field.
On this basis, the possibility arises that some or all of the voltage dependence of proton block from the intracellular side of the membrane may arise from a coupling between K+ in the selectivity filter and H+ in the intracellular vestibule. To what extent such a mechanism contributes to the voltage dependence of proton block will require further experimentation to resolve.
Does Channel Block Arise through the Screening of Surface Charge?
Proton binding to surface charges on lipid membranes can decrease the conductance of some channels by neutralizing (screening) negative surface charges that might normally increase the conductance of the channel by concentrating K+ at the entrance to the pore (Klockner and Isenberg, 1994; Chen et al., 1996
; Benitah et al., 1997
; Morrill and MacKinnon, 1999
). A lipid-screening mechanism would also be consistent with a fast block because screening should not increase the open-channel noise. A lipid-screening mechanism for block of BK channels by protons appears unlikely, however. A recent study by Park et al. (2003)
showed through experiments measuring the kinetics of Ba2+ block of BK channels incorporated into membranes comprised of either neutral or charged lipids that the surface charge on the lipids was too far away from the pore of the channel (due to the large size of the BK channel) to concentrate cations at the entrance to the pore of the channel. Theoretical calculations in the same study supported the experimental conclusion. Thus, if the lipid surface charge is too far away to concentrate K+ at the entrance to the pore, then removing lipid surface charge with protons would have little effect on K+ at the entrance, so the lipid surface charge is not the site of proton block.
The next question is whether surface charges on the protein of the channel itself might be the site of proton block. Glutamate residues frequently are sites of proton block in other channels (Chen et al., 1996; Morrill and MacKinnon, 1999
; Xu et al., 2000
). As a possible candidate for the site of proton block in BK channels, we considered the ring of fixed negative charges formed by eight glutamate residues at the entrance to the intracellular vestibule (Brelidze et al., 2003b
; Nimigean et al., 2003
). This ring of charge doubles the conductance of BK channels by concentrating K+ in the vestibule by an electrostatic mechanism (Brelidze et al., 2003b
). Protonation or screening of the ring of charge by protons might decrease the concentration of K+ at the entrance to the vestibule, decreasing the unitary currents. Support suggesting that protons might act at the ring of charge is the observation in KcsA channels that the introduction of an aspartate residue in the region corresponding to the ring of charge in BK channels significantly increased proton block of unitary KcsA currents (Nimigean et al., 2003
).
In contrast, we found in BK channels that the percentage reduction of unitary currents by proton block at pHi 5.0 was the same in the presence and absence of the ring of charge (Fig. 8 C), indicating that the ring of charge is not the major site of action of proton block for BK channels. This relative lack of effect of the ring of charge on proton block may reflect that the ring of charge would increase the concentrations of both K+ and H+ at the entrance to the intracellular vestibule, with the increased K+ compensating for the effects of the increased H+. The absence of a ring of charge would then decrease the concentrations of both K+ and H+ at the entrance to the intracellular vestibule so that the percentage reduction in currents by protons would be the same with or without the ring of charge at pH 5.0. If protons also bind to the carboxyl residues, in addition to screening, the binding would reduce the effective number of charges in the ring of charge. However, since removing the ring of charge has little effect on proton block (Fig. 8), then partially removing charge from the ring of charge by the binding of protons would also be expected to have little effect on proton block. At pHi 7.0 the percentage reduction of currents due to proton block was slightly less in the presence of the ring of charge than in its absence, suggesting that, if anything, the ring of charge may reduce proton block of BK channels. Thus, the ring of charge is not the site of proton block.
The apparent dissociation constants for proton block of WT BK channels and for mutant BK channels without the ring of charge were 5.1 and 4.9 respectively. Except for the E321 and E324 residues in each of the four subunits that form the ring of charge, there are no apparent candidate residues with a pKa close to 5.0 in the intracellular vestibule of the BK channel pore. It is possible that the site of the proton block is a composite site (oxygen atoms in the selectivity filter?), rather than a site provided by a single residue, or that the surrounding structures change the pKa (Fersht, 1985). Apparent Hill coefficients of 0.290.48 when describing the data with a competitive inhibitory model with a cooperativity factor (Eq. 4) suggests a lack of positive cooperativity in the proton block of the BK channel.
Do Protons Block through an Allosteric Mechanism?
In the previous section it was indicated that the surface charge of the membrane lipids is too far away from the pore of the channel to increase the concentration of K+ or H+ at the entrance to the vestibule. This conclusion does not rule out that the surface charge of the lipids may play some role in proton block. Park et al. (2003) have proposed that the observed decrease in the conductance of BK channels that occurs when negatively charged lipids are replaced with neutral lipids arises from lipid-induced conformational changes of the selectivity filter that depend on the charge of the lipid. On this basis, protons might reduce conductance through a similar allosteric mechanism by changing the properties of the lipids by binding to the negative charges on the lipid head groups. Arguing against such an allosteric mechanism for proton block is that it is difficult to envision how such a mechanism could give rise to the voltage dependence of the block. Furthermore, such an allosteric mechanism for proton block might be expected to reduce unitary current amplitudes for inward, as well as outward, currents, which is not observed (Habartova et al., 1994
). Although if the allosteric mechanism were coupled to depolarization in some manner, then it might not be observed at negative potentials. Protons might also decrease conductance in a different type of allosteric manner by binding to a site within the electric field of the membrane but outside of the pore of the channel. Such an allosteric mechanism could provide voltage-dependent proton block and also competitive inhibition. Whereas direct block of the pore of the channel by protons is a simpler mechanism for proton block, and therefore is to be preferred, our data are not sufficient to exclude more complex allosteric mechanisms.
The Proton Block of BK Channels Contributes to the Sublinearity of Unitary Currents at High Voltages at pH 7.0
We observed that the unitary currents through BK channels increased sublinearly with voltage for depolarizations >100 mV. Our findings indicated that 40% of this sublinearity arose from proton block for data collected at pH 7.0, and became progressively greater for more acidic pHi. Even in the effective absence of protons at pHi 9.0, the i/V plot still deviated from linearity at high voltages, indicating that some factors besides proton block contribute to the sublinearity of currents at the higher voltages. Sublinearity was still observed in solutions that contained no added buffers, or in solutions with chelators to remove trace amounts of Ca2+ or Ba2+, suggesting that some intrinsic property associated with the channel contributes to the sublinearity. This sublinear increase might result from a limitation on the diffusion of K+ from the bulk solution to the intracellular vestibule of the channel (Lauger, 1976
; Andersen, 1983
; Kuo and Hess, 1992
; Hille, 2001
), and/or intrinsic properties of the channel (Yellen, 1984
). These points will be dealt with in greater detail elsewhere.
Although proton block could be appreciable under extreme conditions of low pHi and high voltages, it would be expected that intracellular protons would have little blocking effect on BK channels for physiological voltages and pH. Nevertheless, the marked effect of proton block on unitary currents at the large positive voltages routinely used to investigate mechanisms of selectivity and conductance indicates that proton block needs to be taken into account unless the pH is 8.0.
Limits of K+ Permeation through BK Channels
BK channels have the largest single-channel conductance (250350 pS) of all known K+ channels (Marty, 1981; Pallotta et al., 1981
; Yellen, 1984
; Latorre et al., 1989
; Hille, 2001
). In spite of their large conductance, BK channels are highly selective for K+ over Na+ (Blatz and Magleby, 1984
; Yellen, 1984
; Eisenman et al., 1986
; Latorre et al., 1989
; Hille, 2001
). The largest unitary currents that we observed in this study were
170 pA with 3.4 M [K+]i, 150 mM [K+] outside, a voltage of +250 mV, and a pHi of 9.0. The cord conductance for these conditions was
536 pS at +100 mV. A current of 170 pA requires an average net transfer of
1.1 K+ ions through the channel per nanosecond. The water molecules of the inner sphere surrounding a K+ ion are replaced with a time constant of
1 ns (Diebler et al., 1969
; Hille, 2001
). To enter the selectivity filter most of the hydration shell would have to be removed (Bezanilla and Armstrong, 1972
; Hille, 1973
) by substitution of the carbonyl oxygen atoms within the selectivity filter for the waters of hydration (Zhou et al., 2001
; Hille, 2001
; MacKinnon, 2003
). Thus, the dehydration rate of K+ as it enters the selectivity filter for the largest unitary currents that we observed is similar to the time required for substitution of waters in the inner hydration shell of K+ while K+ is in the bulk solution.
The maximum conductance for the outward currents that we observed, 536 pS, was essentially the same as the maximum theoretical conductance of 550 pS calculated by Berneche and Roux (2003) from the structure of the KcsA channel using a transmembrane potential profile from the open MthK channel (Jiang et al., 2002b
). Yet, for experimental conditions with 100150 mM [K+]i, the unitary currents in BK channels are 34 times higher than those in KcsA channels (Blatz and Magleby, 1984
; LeMasurier et al., 2001
). Why then is the maximum theoretical conductance for KcsA channels the same as the maximum experimentally observed conductance for BK channels? BK channels have a ring of negative charge in the intracellular vestibule that is absent in KcsA channels (Brelidze et al., 2003b
; Nimigean et al., 2003
). This ring of charge doubles the conductance of BK channels for outward currents (Brelidze et al., 2003b
; Nimigean et al., 2003
). The effect of the ring of charge on increasing conductance decreases as the [K+]i is increased, becoming negligible at very high [K+]i (3.4 M), suggesting that the action of the ring of charge is through electrostatic attraction of K+ to the vestibule (Brelidze et al., 2003b
). Because the maximum theoretical conductance for KcsA channels (Berneche and Roux, 2003
) and the maximum experimentally observed conductance for BK channels (Fig. 6) were both obtained with high [K+]i where the effects of the ring of charge would be negligible, then it would be expected that these maximal estimates would be similar for BK and KcsA if these two ion channels have relatively similar conduction pathways except for the ring of charge.
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ACKNOWLEDGMENTS |
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This work was supported in part by grants to K.L. Magleby from the Florida Department of Health Biomedical Research Program, B029, and from the National Institutes of Health, AR32805.
Olaf S. Andersen served as editor.
Submitted: 22 September 2003
Accepted: 2 February 2004
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REFERENCES |
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