Defining the physical gate of a mechanosensitive channel, MscL, by engineering metal-binding sites

Abstract

The mechanosensitive channel of large conductance, MscL, of Escherichia coli is one of the best-studied mechanosensitive proteins. Although the structure of the closed or "nearly-closed" state of the Mycobacterium tuberculosis ortholog has been solved and mechanisms of gating have been proposed, the transition from the closed to the open states remains controversial. Here, we probe the relative position of specific residues predicted to line the pore of MscL in either the closed state or during the closed-to-open transition by engineering single-site histidine substitutions and assessing the ability of Ni2+, Cd2+ or Zn2+ ions to affect channel activity. All residues predicted to be within the pore led to a change in channel threshold pressure, although the direction and extent of this change were dependent upon the mutation and metal used. One of the MscL mutants, L19H, exhibited gating that was inhibited by Cd2+ but stimulated by Ni2+, suggesting that these metals bind to and influence different states of the channel. Together, the results derived from this study support the hypotheses that the crystal structure depicts a "nearly closed" rather than a "fully closed" state of MscL, and that a clockwise rotation of transmembrane domain 1 occurs early in the gating process.

FIGURE 1

R13H MscL confers a GOF growth phenotype in a pH-dependent manner. The E. coli pB104 mscL-null strain transformed with the expression vector pB10b containing either WT or R13H-mutated MscL were grown in citrate-phosphate media at pH 6, pH 7, or pH 8. The growth of bacterial cultures was monitored every 30 min by measuring their optical density (OD₆₀₀). Expression of the channel was induced at minute 120, as indicated by the arrow, by addition of 1 mM IPTG. Bacteria expressing WT MscL showed similar growth rates at all the pHs tested; an average trace is shown.

FIGURE 2

Metal ions increase the pressure threshold of the R13H MscL mutant. WT and R13H-mutated MscL were expressed in the E. coli MJF465 mscS, mscK, and mscL triple-null strain, and the resulting MS channel activities were measured by patch clamp at pH 8.0. Representative traces before (left) and after (right) addition of 5 mM NiCl₂ (top) or CdCl₂ (bottom) solutions to the bath are shown; the upper trace shows channel activity while the lower trace depicts the pressure applied to the patch. Arrows indicate the threshold pressure.

FIGURE 3

Metal binding to the R13H MscL mutant is saturable, reversible, and pH-dependent. (A) The change in the pressure needed to gate WT (triangles) and R13H (diamonds) MscL, as a function of metal ion concentration, is shown. Pressure thresholds for WT and R13H MscL were measured in patch-clamp experiments at increasing concentrations of Ni²⁺ or Cd²⁺ ions applied to the bath at pH 8.0. Error bars are the SEM of three or more independent experiments. (B) The changes in the pressure threshold of R13H MscL in the presence of 5 mM metal ions in the bath, and after wash, are shown (pH 8.0). Values are the ratio between the pressure thresholds ((after/before treatment) − 1), and are expressed as percentages. Error bars are the SEM of four or more independent experiments. (C) The changes in pressure threshold of WT and R13H MscL in the presence of 5 mM metal ions at pH 6.0 (n = 12) and pH 8.0 (n = 7) are shown. Error bars are SEM. *p < 0.05, **p < 0.005 from a paired Student's t-test, control versus treated.

FIGURE 4

Histidine substitutions of residues predicted to be exposed to the lumen lead to channels that show increased threshold in the presence of metal ions; F29H, not predicted to be in the pore, does not. Percent increases in the pressure threshold of WT MscL and TM1 histidine mutants after perfusion of 5 mM Ni²⁺ (top), Cd²⁺ (middle), or Zn²⁺ (bottom) to the bath (pH 8.0) are shown. Error bars are the SEM of five or more independent experiments. *p < 0.01, **p < 0.002 from an unpaired Student's t-test, ratio of mutant (treated/control) versus ratio of wild-type MscL.

FIGURE 5

The affinity of Zn²⁺ binding to histidine mutants suggests that G26, not V23, forms the constriction point in the closed state. (A) The change of the pressure needed to gate WT (solid triangles), V23H (open diamonds), and G26H (shaded circles) MscL, as a function of metal ion concentration, is shown. The pressure thresholds were measured in patch-clamp experiments at increasing concentrations of Zn²⁺ ion applied to the bath. Error bars are the SEM of six or more independent experiments. (B) Percent increases in the pressure threshold of WT (solid), V23H (open), and G26H (shaded) MscL after applying 500 nM (left) or 5 μM (right) Zn²⁺ to the bath (pH 8.0) are shown. Error bars are the SEM of seven or more independent experiments. *p < 0.01 from a paired Student's t-test, control versus treated. A comparison of the ratios (treated/control) at both concentrations also showed a significant difference between G26H versus WT (*p < 0.01 from an unpaired Student's t-test).

FIGURE 6

Ni²⁺ and Cd²⁺ have opposite effects on L19H gating. (A) Representative traces of L19H MscL activities before (left) and after (right) the addition of 5 mM Ni²⁺ or Cd²⁺ solutions to the bath (pH 8.0): the upper trace shows channel activity while the lower trace depicts the pressure applied to the patch. Arrows indicate the threshold pressure. (B) Changes in the pressure threshold of L19H mutant when 5 (left) or 50 (right) mM solutions of Ni²⁺ (shaded) and Cd²⁺ (solid) were added to the bath (pH 8.0). Error bars are the SEM of seven or more independent experiments. *p < 0.01, **p < 0.0005 from a paired Student's t-test, control versus treated.

FIGURE 7

The location of the substituted residues, relative to a proposed constriction point, correlates with the degree of inhibition of channel gating by metal ions. (A) A model of an ideal α-helix showing residues 18–38 of TM1. Residues colored in blue, when substituted with histidine, show the greatest inhibition by metal ions. These are proposed to be the constriction point (G26) and an additional residue (G22), which is slightly clockwise to it (+ direction). A residue colored in green farther clockwise (F29) showed no change in gating properties with either of the metals. Residues colored in red (V23 and I24) show inhibition to a lesser extent. L19H, in orange, shows opposite responses to Ni²⁺ versus Cd²⁺. (B) A model of the E. coli MscL in a closed or nearly closed structure, derived from the M. tuberculosis crystallographic structure, is shown (left). A single subunit (middle) and an enlargement of the relevant region of TM1 (right) are shown. The arrows indicate the predicted clockwise rotation of TM1 upon channel gating, opening (+), and closing (−).