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Published in final edited form as: FEBS Lett. 2010 Feb 6;584(6):1126–1132. doi: 10.1016/j.febslet.2010.02.003

A Molecular Mechanism for Proton-Dependent Gating in KcsA

Luis G Cuello 1, D Marien Cortes 1, Vishwanath Jogini 1, Amornrat Somporpisut 1, Eduardo Perozo 1,2
PMCID: PMC2858269  NIHMSID: NIHMS181782  PMID: 20138880

Abstract

Activation gating in KcsA is elicited by changes in intracellular proton concentration. Thompson et al [1] identified a charge cluster around the inner gate that plays a key role in defining proton activation in KcsA. Here, through functional and spectroscopic approaches, we confirmed the role of this charge cluster and now provide a mechanism of pH-dependent gating. Channel opening is driven by a set of electrostatic interactions that include R117, E120 and E118 at the bottom of TM2 and H25 at the end of TM1. We propose that electrostatic compensation in this charge cluster stabilizes the closed conformation at neutral pH and that its disruption at low pH facilitates the transition to the open conformation by means of helix-helix repulsion.

1. INTRODUCTION

The selective flow of ions across ion channels is regulated through two interrelated gating processes: an energy transduction step where different stimuli (i.e. electric field, ligand binding and membrane stretching) are sensed, and the structural rearrangements that ultimately control the access of ions to the permeation pathway [1–3].

Although a great deal of functional and structural information has been gathered in the last decade regarding KcsA activation and inactivation gating, the nature and location of KcsA pH sensor remains elusive and a number of key questions remain unanswered. Recently, computational and experimental reports have provided specific candidates to the pH-sensor in KcsA. Solution NMR experiments were used to address the protonation state of residues in the intracellular face of C-terminal truncated KcsA [4,5]. Based on observed pH-dependent chemical shifts (but no functional data), it was proposed that H25 at the base of TM1 in KcsA serves the channel proton sensor. In contrast, a recent set of Monte Carlo simulations on the truncated form of KcsA have suggested that protonation of E118 and E120 would promote spontaneous openings of the inner helical bundle [6,7]. Finally, Thompson et al [8] probed the intracellular region of KcsA and provided compelling evidence suggesting that a charge cluster at the C-terminal end of TM2 serves as the pH sensor in KcsA.

In this study, we confirm and extend the findings of Thompson et al [8] in regards to the charged groups involved in proton sensing, and provide additional information on the role of lipids on channel activation. Our data suggests a specific mechanism of action where KcsA proton dependent gating is the consequence of an electrostatic attractive/repulsive balance between a cluster of charges at the intracellular end of both the TM1 and TM2 helices, close to the water/lipid interface.

2. MATERIALS AND METHODS

Molecular biology and biochemical preparation

Single point mutations were introduced in the KcsA wild type DNA sequence by Quickchange protocol, STRATAGENE La Jolla, California. Simultaneous neutralization mutant were made by PCR overlapping methods. Protein expression and purification was carried out as described previously [9].

Spin labeling of cysteine mutants for EPR and reconstitution in liposomes

CW-EPR spectroscopy was carried out as before [10–12]. KcsA cysteine mutants were concentrated to 3–4 mg/ml and labeled with methane-thisulfonate spin-label (MTS-SL; Toronto Research Chemical) in at 10:1 SL to channel molar ratio. Spin-labeled mutant were reconstituted in preformed asolectin or synthetic lipids liposomes in 1:400 channel to lipid molar ratio in a buffer containing 150 mM KCl and 50 mM tris-Cl pH 7.0. X-band CW EPR spectra were obtained in a Bruker ELEXYS spectrometer under the following conditions: 2 mW incident power, 100 kHz modulation frequency and 1 G modulation amplitude.

pH-dependent 86Rb uptake experiments

KcsA WT and mutants were reconstituted in preformed asolectin liposomes containing 200 mM K2SO4 and 20 mM Tris-Cl pH 7.5 (Buffer A) and assayed for electrophoretic 86Rb influx assays as previously described [13], using 10 mM citrate-phosphate buffer and osmolarity adjusted to the internal liposome values with sorbitol. For ion strength dependence experiments, the buffer composition column was adjusted by exchanging sorbitol for isosmolar Choline Chloride.

Cell complementation assay

Potassium uptake deficient E. coli strain LB2003 [14] was used to evaluate the phenotype of KcsA pH-insensitive mutants in vivo. In brief, LB2003 cells were transformed with a KcsA wild type or mutant containing plasmid and the growth rate was evaluated in liquid or solid define media supplemented with 1 mM KCl and 0.5 mM IPTG.

Electrostatics calculations

We used the Adaptive Poisson-Boltzmann Solver (APBS) plugin [15] within VMD [16] to calculate electrostatic potential grid maps using the linearized Poisson Boltzmann equation in a system based on an energy minimized form of KcsA in the closed conformation (1K4C, [17]). In all cases, we have assumed that the ionizable residues exist in their expected, bulk protonation states, since their actual pKa values are not known (and could be quite different to those in bulk). Protein and solvent dielectric constants were set to 2 and 78 respectively. The electrostatic interaction energy between a subunit with the rest of the channel was calculated for WT and open mutant in the closed state using CHARMM [18] PBEQ module. Electrostatic potential maps were displayed using Pymol (http://pymol.sourceforge.net).

3. RESULTS

The charge cluster at the membrane interface of KcsA works as a proton-sensor

Close inspection of the KcsA crystal structure has revealed a cluster of charged residues in spatial proximity at the cytoplasmic ends of TM1 and TM2, very close to the membrane-water interface (Figure 1A, D). We probed the role of these charges as putative components of the KcsA proton-sensor by neutralizing each position and assaying individual mutations by 86Rb-uptake. Figure 1B shows the result of individual cysteine mutagenesis of the charges in TM2 on pH-dependent 86Rb-uptake. Though most of the mutated residues had some influence on KcsA gating, two mutants, R117C and E118C, showed large rightward shifts in the “composite” pKa or EC50 of activation, strongly biasing the closed-open equilibrium to the open conformation (pKa of 7.5±0.1 for R117C and 6.9±0.2 for E118C). Similar results were obtained from individual neutralizations to glutamine (not shown).

Figure 1. A charge cluster near the inner bundle gate is part of the channel pH sensor.

Figure 1

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A, D. Ribbon representation of the intracellular bundle region of KcsA, ionizable residues are shown in licorice representation with basic side chains in blue, acidic side chains in red and histidines in purple. B. 86Rb uptake experiments for individual neutralization mutants in the TM2 cluster. Each pH-dependent uptake experiment was fitted to a Hill equation and activation pKas (EC50) were calculated. Activation pKa: WT=5.89±0.2, R117C=7.46±0.2, E118C=6.83±0.1, E120C=6.2±0.1, R121C=6.47±0.2, R122C=6.41±0.2, H124C=5.89±0.2. C. 86Rb uptake experiments of individual re-introduced charges in a charge-less background mutant lacking the C-terminal domain. Uptake experiments were fitted to a Hill equation and activation pKa were calculated: KcsA=6.18±0.1, R117+=6.94±0.2, E118+=7.98±0.1, E120+=7.69±0.2, R121+=6,74±0.2, R122+=6.39±0.2, H124+=6.56±0.1 and Q-less=6.7±0.2. E. pH-dependent 86Rb uptake experiments of individual neutralization mutants of TM1 N-terminus and amphipatic helix. Data were fitted to a Hill equation and activation pKa were calculated. Activation pKa: Wt=5.69±0.2, R11C=6.47±0.2, K14C=6.38±0.3, H20C=6.41±0.3, H25C=7.59±0.2 and R27C=6.88±0.2. F. Activation pKa for mutations that affect the local electrical charge at position H25. KcsA-wt=5.89±0.2, H25Q=6.9±0.3, H25E=7.7±0.2 and H25R=6.8±0.1.

We tried to reduce the complexity of the cluster by engineering a “charge-less” KcsA, where all charged residues have been neutralized at once (R117Q, E118Q, E120Q, R121Q, R122Q and H124Q). Onto this frame, we reintroduced, one at a time, all the charged residues in the cluster and evaluated their functional effect from 86Rb-uptake experiments. Figure 1C shows that when the two glutamate residues E118 and E120 are uncompensated by the surrounded charges (mutants E118+ and E120+, respectively), the EC50 is severely right-shifted to more basic pH (pKa for E118+ and E120+ are 7.9±0.2 and 7.6±0.3 respectively). Similar behavior was also observed for the arginine residues R117+ and R121+ although less so than with the negative residues.

The second set of charges we evaluated is located at the N-terminal end of TM1 (H25 and R27) and the N-terminal amphipathic interfacial helix (Figure 1D) [19]. Figure 1E shows that the two charged residues located in TM1 generated the largest pKa shifts towards more basic pH: H25C and R27C. These shifts are accompanied by a reduction in the Hill coefficient of the transition, in particular for residue H25, which together with residues R117 and E118 generate the largest functional perturbations in pH dependent gating. Replacing H25 with a isosteric side chain (H25Q, Figure 1F) causes a shift on the activation EC50 to 6.9±0.3 (5.9±0.1 for wild type), a lower pKa side chain (H25E) shifted the EC50 to more basic pH (pKa=7.7±0.2), but when substituted with more basic residue (H25R), pKa for proton-dependent activation gating was shifted to more acidic pH (pKa=6.8±0.1) when compared to H25E substitutions. This seems to suggest that H25 in KcsA helps to stabilize the channel in closed conformation by being fully protonated and therefore positively charged even at neutral pH.

Contribution of the C-terminal domain

Proteolytic cleavage of KcsA C-terminal domain (Fig 2A) has been shown to affect steady state gating behavior by reducing both its apparent cooperativity in 86Rb flux experiments [19] and its open probability at steady state in planar lipid bilayers [20]. These findings led to the suggestion that the C-terminus is required for channel opening and could potentially be the locus for the pH sensor [20]. To investigate this possibility, we have evaluated pH-dependent structural changes at the inner helical bundle by EPR spectroscopy. Fig 2B (top panel) shows that although the C-terminal truncated channel displays a slightly weaker spin-spin dipolar coupling at rest (suggestive of a loose packing in the inner helical bundle), the relative dipolar coupling remains strongly pH-dependent for both KcsA-FL and KcsA-Δ125 in (Figure 2B, bottom). Nevertheless, truncating the C-terminal end generated a significant right-shift in the activation EC50 (pKa for KcsA-FL and KcsA-Δ125 in detergent are 4.1± 0.1 and 5.4± 0.3, respectively) and a reduction in apparent cooperativity when compared to KcsA-FL (Hill number, n for KcsA-FL and KcsA-Δ125 are 1.8 ± 0.2 and 0.7 ± 0.2 respectively) (Figure 2C). Taken together, these results indicate that while its presence tends to stabilize the inner gate in its closed conformation [19,21], the C-terminal helical bundle only modulates the channel pH-sensor and does not play a direct role in channel activation.

Figure 2. The C-terminal helix bundle is not the pH sensor.

Figure 2

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A. Graphical depiction of KcsA full-length channel and C-terminal domain truncated by proteolytic treatment with chymotrypsin. B. CW EPR spectra of spin labeled mutant KcsA-G116C illustrates the structural consequence of truncating the c-terminal domain. Top, The bundle gate is slightly opened by C-terminal truncation: EPR spectra at pH 7.0 for KcsA full-length (black trace) vs. KcsA Δ125 (red trace). Bottom, The bundle gate undergoes pH-dependent conformational changes in the absence of the C-terminal helix bundle: pH-dependent EPR spectra changes of KcsA full-length pH 8.0 (black trace) vs. KcsA-Δ125, pH 3.0 (red trace). C. pH-dependence of the conformational changes at the activation gate monitored by EPR spectroscopy: full length-KcsA (black trace, EC50=4.1±0.1), KcsA-Δ125 (red trace, EC50=5.4±0.3).

Influence of ionic strength and the electrostatic basis of proton-dependent gating

The time course of KcsA-catalyzed 86Rb uptake at high (200 mM NaCl) and low (5 mM NaCl) ion strength (S) (pH 4.0) is shown in Figure 3A. Lowering S leads to a dramatic increase in 86Rb uptake, an empirical observation reported early on [22]. Systematically changing the intracellular electrolyte concentration revealed an asymptotic dependence of 86Rb uptake with an apparent mid-point near 20 mM, leveling off beyond 100 mM NaCl, (Fig. 3B). This behavior is well fit by the Debye-Huckel limiting law (Fig. 3B, black line), according to: 1/κ = (εε0RT/F2)0.5 S−0.5, where the ion strength S=Σcz2/2, being c the concentration and z the charge of the ionic species in the electrolyte[1,23]. Modifying the local electrical field by changing the ionic strength (S) is predicted to modulate the effectiveness of electrostatic repulsion/attraction and thus affect the magnitude of the pH dependent activity [24–26]. The ionic strength effects on KcsA steady state activity were recapitulated at the single channel level (Fig. 3C), and are characterized by increases in the mean open time and the frequency and length of single channel bursts.

Figure 3. Intracellular ionic strength modulates KcsA open probability and conformation of the activation gate.

Figure 3

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A. Time-dependent 86Rb+ uptake under conditions of high (■, 200 mM NaCl) and low (○, 400 mM sorbitol) ionic strength. In both cases, the solution facing the intracellular side was buffered at pH 4. B. Ion strength dependence of the magnitude of the steady-state 86Rb+ uptake. C. Left, representative single channel recordings of KcsA in symmetric 200 mM KCl or in 200 mM/1 mM KCl (extracellular/intracellular) asymmetric solutions. The right panels show the corresponding open time histograms. Data were obtained at −100 mV, filtered at 2 KHz and were fitted to a sum of two expoenential: 1.21.27 and 6.51 ms in high ionic strength; 2.6 and 7.39 ms in low ionic strength. D. Upper panel, CW-EPR spectra from mutants reporting the conformation of the intracellular gate (T112C and G116C). Data were obtained close to the EC50 of activation (pH 4.5) under conditions of high (400 mM) and medium (50 mM) and low (5mM) ionic strength. Spectra are shown normalized to the total number of spins in the sample. Lower panel, Ion strength dependence of the conformational changes at positions 112 and 116 (filled circles).

The influence of S on the conformation of the activation gate was evaluated in KcsA spin-labeled at two different positions along TM2 (T112C and G116C), near the EC50 of activation (pH ~ 4.5, to maximize the effect of S). Figure 3D (top) shows representative EPR spectra at position 116 in 5, 50 and 400 mM NaCl at the intracellular side of the channel. Under partially activated conditions and low ionic strength, the lower or activation gate undergoes large movements towards the open conformation, but mostly samples the closed state at 400 mM NaCl (no major spectral changes). Similar results were obtained by monitoring the extent of dipolar coupling at position T112 (Figure 3D bottom). Overall, these results suggest, rather convincingly, that an electrostatic process mediates proton dependent gating in KcsA.

To qualitatively illustrate this point, we used Adaptive Poisson-Boltzmann Solver (APBS) [15] to calculate the electrostatic surface potential at the KcsA inner helical bundle. This was carried out on WT KcsA with protonation of titratable residues corresponding to pH 7.0 and 3.0. Figure 4 displays slices through the calculated isopotential contours at ± 1kT/e (~25 mV) in 150 mM KCl, shown in a side view perpendicular to the plane of the membrane and as a cut through the core of the inner gate/pH sensor (dotted line). At pH 7.0, a large positive potential generated by arginine and histidine residues is partially compensated by the negative potential from glutamate and aspartate residues intertwined at the subunit interface (Figure 4, left). Protonation of acidic residues (E118 and E120) to simulate acidic pH leads to a large reduction of negative potential in the bundle, creating a large positive potential, which we submit, would be large enough to open the channel by electrostatic repulsion (Figure 4, right), as shown in an engineered constitutively open KcsA mutant [27] and similar to Thompson et al. set of mutants H25R/E118A/E120A [8].

Figure 4. Isopotential surface calculation at the KcsA inner helical bundle illustrates the electrostatic nature of KcsA pH sensor.

Figure 4

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Graphical representation of the Poisson-Boltzmann equation in 150 mM KCl with calculated isopotential contours at 1kT/e (~25mV). Left-panel, KcsA-wt at pH 7; right-panel, KcsA-Wt at pH 3.

4. DISCUSSION

The molecular basis of proton sensitivity has typically been associated with simple protonation-deprotonation reactions of ionizable groups. This has been the case in a variety of potassium, TRP and chloride channels [28,29] and in ASIC channels, where gating appears to be the result of the interplay between protonation and Ca++ binding [30]. In KcsA, the fact that there are more than thirty ionizable side chains per monomer on the intracellular side of the channel have posed an important challenge in defining the nature of the pH sensor.

As a first approximation, the C-terminal domain, a densely charged helical bundle that projects into the cytoplasm [19] appeared to be the logical candidate. Indeed, the difficulties of recording single channel activity from a truncated synthetic form of KcsA led to the suggestion that the channel cannot open in the absence of the C-terminal domain [20,31]. However, our functional experiments have shown that C-terminal truncated KcsA is still proton activated [19] and that in the absence of the last 35 C-terminal residues, the gate opens fully and its conformational rearrangement is essentially indistinguishable from that of the full-length channels, as monitored by spin-spin dipolar coupling (Fig 2B and 2C).

Three recent reports have made specific proposals for the molecular nature of the pH sensor in KcsA. Tacheuchi et al [5]., have carried out a solution NMR analysis of KcsA in detergent micelles, evaluating pH dependent changes by 1H15N-TROSY HSQC experiments. They find that the main pH-dependent rearrangements take place around the inner helix bundle and that neutralizing residue H25, which they suggest is the pH sensor, can abolish these changes. Independently, Miloshevsky and Jordan have carried out a computational analysis of KcsA gating based on Monte Carlo simulations inner helix bundle movements [6,7]. They propose that E118-E120 pair in TM2 forms the pH sensor in KcsA, and that protonation of both residues is required for channel opening. Finally, Thompsom et al [8] have identified the charge cluster surrounding TM2 as the pH sensor in KcsA, suggesting a gating mechanism actuated by the disruption of a network of salt bridges/hydrogen bonds upon protonation.

Our present findings are incompatible with models based on relative simple protonation reactions at a single or double site. Instead, systematic mutagenesis of all ionizable side chains in this cluster (Fig 5A) revealed several residues critical for proton-sensitivity: H25 and R27 at the start of TM1, and R117, E118, E120 and R121 at the base of TM2. But in particular, neutralization of H25, R117 or E118 generates the largest shifts in EC50 and pH-dependent slope among the members of this cluster (Fig. 1). These residues have also been identified as ‘gain-of-function’ mutations in random mutagenesis studies [32]. Key members of this charge cluster are positioned within 5 Å of each other, pairs E118-R121 and E118-R122 in the same subunit, and more importantly, pairs H25-E118 and E120-R121 in neighboring subunits (Fig 5B). Interestingly, although our neutralization mutagenesis suggests that the R117-E118 influence each other, the distances between the R117-E118 pairs are 10.5 Å and 9.8 Å, inter- and intra-subunit, respectively. These exceed the minimal distance required for salt bridge formation (≤ 4 Å [33]) and thus make it unlikely that these charged residues are forming an ion-pair in the closed conformation. Our proposal for a pH sensor in KcsA matches that suggested by Thompson et al [8]. But while Thompsom et al point to the importance of the stability of the salt bridge and hydrogen-bonding network, the present work highlights the role of electrostatic repulsion and local Debye-length modulation in triggering KcsA activation gating.

Figure 5. A mechanism for proton-dependent activation gating at the KcsA inner helical bundle.

Figure 5

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A. 3D model of the KcsA proton sensor at the inner helical bundle. Upper panel, view from the plane of the membrane; lower panel, intracellular view. Helices are represented as ribbons and relevant side chains in licorice representation B. Putative map of proposed interacting partners with their distances in A. Two adjacent subunits are shown. C. Cartoon model representing the basic principle underlying proton activation in KcsA. At pH 7 (closed state, left), the electrical fields generated by opposite charged residues balance each other stabilizing the closed conformation. At pH 4, protonation of the negatively charged side chains (E118 and E120) results in a net positive electrostatic field, leading to helix-helix repulsion and the stabilization of the open conformation.

The reduction of KcsA channel open probability at high ionic strengths is likely a consequence of the shielding/screening effect of the electrolyte solution on the charged residues responsible for the large local electric field that lead to helix repulsion and gate opening, as quantitatively described by the Debye-Huckel limiting law (Fig 3). This is clearly shown from the dependence of the dipolar coupling at positions 112 and 116 (which indicates inter-subunit helix proximity) with the ion strength of the intracellular milieu. Therefore, a reduction in the effective Debye length at high salt concentrations will be accompanied by a reduction in the effectiveness of the local electric fields, decreasing the extent of helix-helix repulsion at the inner gate.

We suggest that the members of the charge cluster are arranged so that the negative electric field around E118 and E120 at neutral pH is somewhat balanced by the surrounding positively charged residues, H25, R27, R117 and R121 (Fig 4). The electrostatic compensation among side chains stabilizes the activation gate in the closed conformation at neutral pH, and disruption of this delicate electrostatic balance facilitates the transition to the open conformation. This suggestion is in agreement with the observation that most of our mutations in the charge cluster biased the closed-open equilibrium towards the open conformation. We propose that at low pH, E118 and E120 become protonated, generating a strong electrostatic repulsion between the positively charged residues H25, R27, R117 and R121, leading to the opening of the activation gate and the onset of ion conduction (Fig 5C). This electrostatic repulsion mechanism would predict that de-protonating positive residues by increasing pH above their pKa (~12.48 for Arginines), or by neutralizing them via mutagenesis should in principle open the proton-dependent gate of KcsA, as clearly shown in the behavior of a constitutively open mutant found by Thompson et al [8], as well as a constitutively open mutant designed with opposite charge polarity [27].

Acknowledgments

We thank Drs. B. Roux H Mchaourab and R. Nakamoto for helpful discussions and comments on the manuscript. S. Chakrapani, J. Cordero-Morales, O. Dalmas, J Santos and the members of the Perozo lab for experimental advice and comments on the manuscript. This work was supported in part by NIH grant R01-GM57846 and by a gift from the Palmer family.

Footnotes

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