Functional design of bacterial mechanosensitive channels. Comparisons and contrasts illuminated by random mutagenesis.
MscS and MscL are mechanosensitive channels found in bacterial plasma membranes that open large pores in response to membrane tension. These channels function to alleviate excess cell turgor invoked by rapid osmotic downshock. Although much is known of the structure and molecular mechanisms underlying MscL, genes correlating with MscS activity have only recently been identified. Previously, it was shown that eliminating the expression of Escherichia coli yggB removed a major portion of MscS activity. YggB is distinct from MscL by having no obvious structural similarity. Here we have reconstituted purified YggB in proteoliposomes and have successfully detected MscS channel activity, confirming that purified YggB protein encodes MscS activity. Additionally, to define functional regions of the channel protein, we have randomly mutagenized the structural gene and isolated a mutant that evokes a gain-of-function phenotype. Physiological experiments demonstrate that the mutated channel allows leakage of solutes from the cell, suggesting inappropriate channel opening. Interestingly, this mutation is analogous in position and character to mutations yielding a similar phenotype in MscL. Hence, although MscS and MscL mechanosensitive channels are structurally quite distinct, there may be analogies in their gating mechanisms.
Okada
Kuniyuki
K
Department of Physiology, University of Texas Southwestern Medical Center, Dallas, Texas 75235-9040, USA.
Moe
Paul C
PC
Blount
Paul
P
D001426:Bacterial Proteins / Q000502:physiology; D002462:Cell Membrane / Q000502:physiology; D017931:DNA Primers; D004926:Escherichia coli / Q000254:growth & development / Q000502:physiology*; D029968:Escherichia coli Proteins / Q000235:genetics / Q000502:physiology*; D015964:Gene Expression Regulation, Bacterial; D006863:Hydrogen-Ion Concentration; D007473:Ion Channels / Q000235:genetics / Q000502:physiology*; D016296:Mutagenesis; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000378:metabolism; D017384:Sequence Deletion
D001426:Bacterial Proteins; D017931:DNA Primers; D029968:Escherichia coli Proteins; D007473:Ion Channels; C086596:MscL protein, E coli; C469542:MscS protein, E coli; D011994:Recombinant Proteins