Single streptomyces lividans K(+) channels: functional asymmetries and sidedness of proton activation

Abstract

Basic electrophysiological properties of the KcsA K(+) channel were examined in planar lipid bilayer membranes. The channel displays open-state rectification and weakly voltage-dependent gating. Tetraethylammonium blocking affinity depends on the side of the bilayer to which the blocker is added. Addition of Na(+) to the trans chamber causes block of open-channel current, while addition to the cis side has no effect. Most striking is the activation of KcsA by protons; channel activity is observed only when the trans bilayer chamber is at low pH. To ascertain which side of the channel faces which chamber, residues with structurally known locations were mapped to defined sides of the bilayer. Mutation of Y82, an external residue, results in changes in tetraethylammonium affinity exclusively from the cis side. Channels with cysteine residues substituted at externally exposed Y82 or internally exposed Q119 are functionally modified by methanethiosulfonate reagents from the cis or trans chambers, respectively. Block by charybdotoxin, known to bind to the channel's external mouth, is observed only when the toxin is added to the cis side of channels mutated to be toxin sensitive. These results demonstrate unambiguously that the protonation sites linked to gating are on the intracellular portion of the KcsA protein.

Figure 3

Currents through single KcsA channels. Recordings of single KcsA channels were made under conditions of Fig. 1. (A) Representative channel openings at the indicated voltages. (B) Open-channel current–voltage relation. Standard errors are smaller than the width of the points. Solid curve has no theoretical meaning.

Figure 1

Voltage-dependent gating of KcsA. A membrane containing at least five KcsA channels was held at 175 mV, with 100K7//100K4 solutions (cis//trans). Voltage was reversed in polarity where indicated. The current record was blanked during amplifier saturation after the voltage change and was subsequently corrected by fitting blank capacitative transients. Dashed lines represent closed-state level in all figures.

Figure 2

pH dependence of KcsA. Currents were recorded at 200 mV in symmetrical 100-mM K⁺ solutions, with pH variation on cis and trans sides, as indicated. Channels were first recorded with pH 4 on both sides. The cis chamber was brought to pH 7, and more records were collected. After perfusion to pH 7 trans, where no openings were observed, the trans chamber was returned to pH 4 to confirm reversibility.

Figure 9

Effect of MTSET on TEA sensitivity of KcsA-Y82C. Time line (top) indicates the sequence of addition of TEA (10 mM) and MTSET (350 μM). Currents were recorded from Y82C at 200 mV. All-points amplitude histograms were constructed from 30–40 s of continuous data. The data shown are from a single experiment; the experiment was repeated two more times with similar results.

Figure 4

TEA blockade of KcsA. (Top) KcsA channels were recorded at 200 mV with indicated TEA concentrations added to the 100-mM K⁺ solutions; these data were filtered at 1 kHz. (Bottom) Open-channel current, i, normalized to the value without TEA, i₀, is shown with single-site inhibition curves, with K _i = 3.2 and 22 mM for cis and trans, respectively.

Figure 5

Block of single KcsA channels by trans Na⁺. Single KcsA openings were recorded in 100 mM K⁺ solutions with or without Na⁺ added to either side. (Left) Raw current traces in the absence of Na⁺ (control) or with 10 mM Na⁺ added to the trans side. (Right) Open-channel I–V curves. ○, no Na⁺ added; •, 30 mM Na⁺ cis; ▪, 10 mM Na⁺ trans. Solid curve through the control points is a spline fit with no theoretical significance. Solid curve through the trans Na⁺ points is the prediction of a single-site Woodhull 1973 blocking mechanism, with an effective valence of 0.5 and a zero-voltage dissociation constant for Na⁺ of 322 mM.

Figure 6

Locations of selected residues on KcsA structure. For clarity, only two KcsA subunits are shown in this RASMOL image. Space-filled residues are as follows. Violet, W26-Y45-W87-W113: markers of the lipid polar headgroup region. Yellow, Y82: position influencing cis TEA block. Green, Q58-T61-R64: positions mutated to strengthen CTX binding. Cyan, Q119: position modified by trans MTSES when mutated to cysteine.

Figure 7

Sidedness of aromatic TEA blocking site. TEA inhibition of KcsA channels substituted at position 82 was assessed as in Fig. 4. (A) cis TEA block. Solid curves are Langmuir fits with K _i of 3.2, 23, and 143 for 82Y (▪), 82C (▴), and 82T (•) channels, respectively. (B) Trans TEA block. Solid curve is Langmuir function with K _i = 25 mM.

Figure 8

MTSET modification of KcsA-Y82C. Single Y82C channel openings were recorded in symmetrical 100 mM K⁺. After recording under control conditions, MTSET (70 μM) was added to the cis chamber. Currents were monitored at negative potentials until an obvious change in conductance was observed (∼7 min). Unreacted MTSET was then removed by perfusion, and the “+MTSET” recordings were collected. (Top) Raw channel records. (Bottom) Open-channel I–V curves.

Figure 11

MTSES modification of KcsA-Q119C. Open-channel I–V curves were determined on the Q119C mutant before and after addition to MTSES to the trans chamber. After collecting control records, the trans chamber was perfused with 100K7 solution containing 200 μM MTSES, and the reaction was allowed to proceed for 2 min. Fresh 100K4 solution without MTSES was then reintroduced, and the I–V curve was recorded. Data shown in the figure are from a single experiment; similar results were obtained in three separate bilayers.

Figure 10

CTX block of KcsA. KcsA wild-type or KcsA-Tx triple mutant channels were held at 150 mV in 100 mM K⁺ solutions; bovine serum albumin (50 μg/ml) was also included in the cis solution to suppress nonspecific binding of CTX to the chamber walls. (A) Records are displayed before (control) or after (+CTX) addition of 1 μM CTX to the cis chamber. (Top) Wild-type KcsA; (bottom) KcsA-Tx. (B) Representative open-time histograms of KcsA-Tx collected from 30 s of continuous data for each condition indicated. Time constants were determined from single-exponential fits to the histograms.