Structural and functional differences between two homologous mechanosensitive channels of Methanococcus jannaschii

Abstract

We report the molecular cloning and characterization of MscMJLR, a second type of mechanosensitive (MS) channel found in the archaeon Methanococcus jannaschii. MscMJLR is structurally very similar to MscMJ, the MS channel of M.jannaschii that was identified and cloned first by using the TM1 domain of Escherichia coli MscL as a genetic probe. Although it shares 44% amino acid sequence identity and similar cation selectivity with MscMJ, MscMJLR exhibits other major functional differences. The conductance of MscMJLR of approximately 2 nS is approximately 7-fold larger than the conductance of MscMJ and rectifies with voltage. The channel requires approximately 18 kT for activation, which is three times the amount of energy required to activate MscMJ, but is comparable to the activation energy of Eco-MSCL: Our study indicates that a multiplicity of conductance-wise and energetically well-tuned MS channels in microbial cell membranes may provide for cell survival by the sequential opening of the channels upon challenge with different osmotic cues.

Fig. 1. Sequence alignment of MscMJLR and MscMJ. The alignment revealed 44% sequence identity. Identical residues are marked with asterisks. The highly conserved C-terminal cluster of charged residues is boxed.

Fig. 2. Structural similarities and differences of the two MS channels of M.jannaschii. (A) Hydropathy profile of MscMJLR (black line) and MscMJ (red line) and predicted helical regions (black bars). Positive numbers indicate relative hydrophobicity of the protein profile. (B and C) Helical wheel representation of the putative TM1 domains of MscMJLR and MscMJ, respectively. Charged residues are boxed and the start of each helix is marked with an asterisk. Arrows indicate positions at which hydrophobic residues within the TM1 helix of MscMJLR are replaced with hydrophilic (charged) residues in the MscMJ TM1 helix.

Fig. 3. (A and B) Number of transmembrane regions predicted for MscMJLR and MscMJ, respectively, obtained by the TMHMM detection program (see Materials and methods). Five TM helices were predicted for both proteins. (C) Prediction of the coiled-coil regions for MscMJLR and MscMJ. Two regions with a high probability of adopting coiled-coil conformation were identified in MscMJLR, whereas there was no such conformation in the corresponding sequence of MscMJ. (D) Prediction of the coiled-coil regions for Eco-MscL revealed two regions with coiled-coil conformation similar to MscMJLR.

Fig. 4. Activity of the recombinant MscMJLR. (A, left) SDS–PAGE of the Ni-NTA purified His₆ MscMJLR protein expressed in E.coli. Lane 1, total E.coli proteins before induction with IPTG; lane 2, total E.coli proteins after induction with IPTG; lane 3, purified and concentrated MscMJLR protein. (A, right) A current trace and the corresponding pressure trace of the liposome reconstituted MscMJLR activated by suction applied to the patch pipette at a pipette potential of +30 mV. C, closed state of channel; O1, open state of channel. (B, left) Current–voltage relationship for MscMJLR in a symmetric recording buffer, containing 200 mM KCl, 40 mM MgCl₂ and 5 mM HEPES pH 7.2, rectifies with voltage. The I–V plot was fitted by a second order polynomial function. Each data point is presented as a mean ± SE (n ≥ 5). Except for the data point obtained at +70 mV error bars are smaller than the filled circles. (C) Current traces of MscMJ showing difference in the size of the single-channel current recorded at +50 and –50 mV pipette potential. The negative pressure applied to the patch pipette was 55 and 35 mmHg, respectively. C, closed state of channel; O1, open state of channel.

Fig. 5. Comparison of conductive and MS properties of the two MS channels of M.jannaschii. Current–voltage relationship for (A) MscMJLR and (B) MscMJ obtained in a symmetric recording solution of 200 mM KCl, 5 mM MgCl₂, 5 mM HEPES pH 7.2 (closed symbols) and after replacing the bath solution with a 3-fold gradient of 600 mM KCl, 5 mM MgCl₂ and 5 mM HEPES pH 7.2 (open symbols). The reversal potential was obtained for the same patch by fitting a second order polynomial curve to the current–voltage plots. The calculated reversal potentials for potassium E_K (+28 mV) and chloride E_Cl (–27 mV) are marked by arrows. The open probability of (C) MscMJLR (n = 5) and (D) MscMJ (n = 4) plotted versus negative pressure applied to the patch pipette was fitted to the Boltzmann distribution function of the form NP_o = NP_o^max [1 + exp α(p_1/2 – p)], where NP_o and NP_o^max are the open probability and the maximum open probability, respectively, N is the number of channels in a liposome patch, α is the sensitivity to pressure corresponding to the slope of the linear fit of the function ln [NP_o/(NP_o^max – NP_o)], p is the negative pressure applied to the patch pipette and p_1/2 is the negative pressure at which P_o = 0.5. For a summary of the Boltzmann characteristics of MscMJLR and MscMJ compared with other prokaryotic MS channels see Table I. (B) and (D) were adapted from Kloda and Martinac (2001a).