Activation-coupled inactivation in the bacterial potassium channel KcsA

Abstract

X-ray structures of the bacterial K+ channel KcsA have led to unparalleled progress in our understanding of ion channel structures. The KcsA channel has therefore been a prototypic model used to study the structural basis of ion channel function, including the gating mechanism. This channel was previously found to close at near-neutral intracellular pH (pH(i)) and to open at acidic pH(i). Here, we report the presence of a previously unknown channel inactivation process that occurs after the KcsA channel is activated. In our experiments, mammalian cells transfected with a codon-optimized synthetic gene encoding the KcsA protein expressed K+-selective channels that activated in response to a decrease in pH(i). Using patch-clamp and rapid solution exchange techniques, we observed that the KcsA channels inactivated within hundreds of milliseconds after channel activation. At all tested pHs, inactivation always accompanied activation, and it was profoundly accelerated in the same pH range at which activation increased steeply. Recovery from inactivation was observed, and its extent depended on the pH(i) and the amount of time that the channel was inactive. KcsA channel inactivation can be described by a kinetic model in which pH(i) controls inactivation through pH-dependent activation. This heretofore-undocumented inactivation process increases the complexity of KcsA channel function, but it also offers a potential model for studying the structural correspondence of ion channel inactivation.

Fig. 1.

Inactivation of KcsA channels expressed in COS-1 cells. (A) Confocal micrograph of untransfected COS-1 cells (Left) giving no green fluorescence (Right). (Scale bar: 50 μm.) (B) Effect of an acidic pH_i pulse on the current in a patch excised from an untransfected COS-1 cell. (C) Confocal micrograph of COS-1 cells expressing fusion KcsA-EGFP proteins that emit green fluorescence. (D) Representative macroscopic current of KcsA channels. An acidic pH_i pulse (pH_i = 3.4) was applied during the period marked by the bar. Baseline shift at the acidic pH_i was subtracted from the current trace. Inset shows the peak current phase on an expanded time scale, in which the red line superimposed on the current trace represents the sum of three exponentials with time constants of 25 ms, 202 ms, and 1.3 s, respectively. The dotted lines crossing the current traces indicate zero current. (E) pH_i dependence of the time constants and relative amplitudes of inactivation exponential components (n = 2–11). A_s/A_f is the ratio of the amplitude of the slower inactivation component to the amplitude of the faster inactivation component. Here and in Figs. 2 and 3, the dotted lines marked “model” represent predictions of the kinetic model that is formulated in Fig. 5A.

Fig. 2.

pH_i-dependent recovery from inactivation and pH_i-dependent activation. (A) Superimposed macroscopic KcsA channel currents show recovery from inactivation in response to a series of two acidic pulses (shown in Inset). Each current trace is marked in a different color, and all three current traces were recorded in the same membrane patch. The current traces have been scaled to the peak currents at the conditioning pulses. The dotted line indicates zero current. (B) Statistical presentation of the recovery from inactivation (n = 5–7). The relative peak current is the ratio of the peak current at the test pulse to that at the conditioning pulse. (C) Macroscopic KcsA channel currents show activation and irreversible inactivation components in response to three successive acidic pulses (indicated by bars). (D) pH_i dependence of the irreversible inactivation component. The measured peak currents (indicated by “c” in C) after 50-s conditioning pulses at a test pH_i were normalized to the control peak currents (indicated by “a” in C) in response to a 300-ms pulse of pH_i 3.2. n = 3–7. (E) Relationship between pH_i and peak current. The peak currents at given pH_i levels (as indicated by “b” in C) were normalized to the control peak currents (indicated by “a” in C).

Fig. 3.

Gating kinetics of single KcsA channels. (A) An example of multiple KcsA channel currents demonstrating the relationship between the macroscopic and single-channel KcsA currents. (Left) Multiple KcsA channel current elicited by an acidic pH_i pulse in a patch excised from a COS-1 cell expressing KcsA-EGFP. (Right) The superimposed currents through three open channels are shown on an expanded time scale (taken from the time marked by the triangle in Left). Multiple-channel current levels anticipated from the single-channel current amplitude are indicated either by a column of bars (in Left) or long-dashed lines together with the numbers of open channels (in Right). (B) Representative single KcsA channel currents recorded in a patch containing a single active channel. Right shows the current trace on an expanded time scale. In A and B, the dotted lines indicate zero current. (C) Corresponding histograms of open times (Left) and closed times (Right) fitted with a single exponential and three exponentials, respectively. (D) Plot of the overall rate constant leaving the open state (the reciprocal of mean open time) against the pH_i.

Fig. 4.

Ionic selectivity of single KcsA channels. (A) Representative responses of single KcsA channels to the shown voltage-ramp protocol at a pH_i level of 7.4 (Top) and at a pH_i level of 3.5 in the presence of various ionic concentrations (Middle and Bottom). 150/10, 150 mM intracellular K⁺, 10 mM extracellular K⁺, 140 mM extracellular Na⁺, and 150 mM symmetric Cl^–; 150/150, in the presence of 150 mM symmetric KCl; 10/150, 10 mM intracellular K⁺, 150 mM extracellular K⁺, 140 mM intracellular Na⁺, and 150 mM symmetric Cl^–. The current trace in Top (pH_i of 7.4) and the current trace marked 150/10 were recorded successively from the same patch. The panel marked 10/150 contains superimposed current traces. The dotted lines in the current crossing the traces indicate zero current. (B) I–V plots show reversal potentials under the ionic conditions corresponding to those indicated in A. Each data point (n = 4–10) of the single-channel current amplitude in the I–V plots is the mean value of the maximal current amplitudes measured within a 5-mV window. The continuous lines represent cubic regressions fit to the data points.

Fig. 5.

Kinetic modeling of the KcsA channel gating process. (A) Scheme for the kinetic model of KcsA channel gating. The scheme models a channel composed of four identical subunits; each has a resting, closed state (R), a protonated, activated state (H⁺A), and a protonated, closed state (H⁺C). The channel opens only when all subunits are activated, which is denoted by 4(H⁺A). The H⁺ binding transition (R–H⁺A) of an individual subunit occurs independently, whereas the transition between H⁺A and H⁺C involves cooperativity between subunits. The model channel can also undergo a concerted move to the irreversible inactivated state 4(H⁺I). Stable dwelling in the irreversible state is assumed to be independent of the protonation status of the subunits (symbolized by shaded H⁺). After being rounded to the nearest representable values, the estimated rate constants and cooperativity factor are k = 1,600,000 s^–1·M^–1, k′= 100 s^–1, α = 1.75 s^–1, β = 1.25 s^–1, and χ = 0.5. [H⁺] is the proton concentration. (B) Simulated macroscopic currents (the three top current traces) of KcsA channels and the macroscopic behavior of the model (the bottom trace) in response to an acidic pH_i pulse. Inset shows the peak current phase of the current trace below it on the expanded time scale. The macroscopic currents and the model behavior were calculated by using the sim program of the qub suite. A total of 100 channels having a uniform closed level and a uniform open level were included in the current simulation. Noises with SD of 10–15% of the single-channel amplitude were added to the closed and open channel levels. The simulation reconstitutes the macroscopic current shown in Fig. 1D under the same conditions used for recording the current. The dotted lines indicate zero current. (C) Model-predicted effect of prolonged exposure to low pH_i on pH_i–activity relationship. KcsA channel activity is represented by open probability, which was measured from computed channel activities at 50 and 200 s after an application of low pH_i started.