Bacterial mechanosensitive channels MscS and MscL directly respond to tension in the cytoplasmic membrane. It appears not to be a coincidence that both channels are anchored to membrane lipids with positively charged residues primarily at the cytoplasmic interface of the inner membrane, leaving the periplasmic side of the barrel the freedom to move and tilt. The functional cycle of MscS involving opening ↔ closing and inactivation ↔ recovery transitions relies on two prominent anchors, R46 and R74, that reside at the level of cytoplasmic phosphates. WT MscS exhibits voltage-dependent inactivation and recovery, showing deeper inactivation and slower recovery under depolarizing voltages (+60 mV) compared to hyperpolarizations (−60 mV). Substitutions of both R46 and R74 with poor interfacial anchors (A or D) result in no expression, whereas G, I, L, or V substitutions express but produce non-functional channels. Patch-clamp experiments showed that polar substitutions in positions 46 and 74, which maintain H-bonding capability to lipid phosphate groups (N, Q, S), preserve expression, activation, voltage-dependence of inactivation and recovery, as well as osmotic rescuing. Aromatic (W, Y) substitutions produce channels that completely lack voltage sensitivity of inactivation and recovery, but are otherwise functional. Insertion of periplasmic anchors (R or W) in flexible N-terminal segments (positions 3-21) did not produce strong phenotypes, however, the A22R substitution, which faces outer-leaflet phosphates, abolishes inactivation without affecting opening. These data show that the inactivation and recovery transitions are sensitive to the polarity of the cytoplasmic ends of TM1 and TM2 and are likely associated with the change of hydration near these segments which can be altered by voltage. The insertion of a periplasmic anchor (R22) restrains the position of the TM1-TM2 pair of helices, which require a higher degree of freedom during inactivation.
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