Electrical and G-protein Regulation of CaV2.2 (N-type) Channels.

How G-proteins inhibit N-type, voltage-gated, calcium-selective channels (Ca V 2.2) during presynaptic inhibition is a decades-old question. G-proteins Gßγ bind to intracellular Ca V 2.2 regions, but the inhibition is voltage-dependent. Using the hybrid electrophysiological and optical approach voltage-clamp fluorometry, we show that Gßγ acts by selectively inhibiting a subset of the four different Ca V 2.2 voltage-sensor domains (VSDs I-IV). During regular "willing" gating, VSDs I and IV activation resemble pore opening, VSD III activation is hyperpolarized, and VSD II appears unresponsive to depolarization. In the presence of Gßγ, Ca V 2.2 gating is "reluctant": pore opening and VSD-I activation are strongly and proportionally inhibited, VSD IV is modestly inhibited while VSD III is not. We propose that Gßγ inhibition of VSD-I and -IV underlies reluctant Ca V 2.2 gating and subsequent presynaptic inhibition.

Release of the type I secreted alpha-haemolysin via outer membrane vesicles from Escherichia coli.

The alpha-haemolysin is an important virulence factor commonly expressed by extraintestinal pathogenic Escherichia coli. The secretion of the alpha-haemolysin is mediated by the type I secretion system and the toxin reaches the extracellular space without the formation of periplasmic intermediates presumably in a soluble form. Surprisingly, we found that a fraction of this type I secreted protein is located within outer membrane vesicles (OMVs) that are released by the bacteria. The alpha-haemolysin appeared very tightly associated with the OMVs as judged by dissociation assays and proteinase susceptibility tests. The alpha-haemolysin in OMVs was cytotoxically active and caused lysis of red blood cells. The OMVs containing the alpha-haemolysin were distinct from the OMVs not containing alpha-haemolysin, showing a lower density. Furthermore, they differed in protein composition and one component of the type I secretion system, the TolC protein, was found in the lower density vesicles. Studies of natural isolates of E. coli demonstrated that the localization of alpha-haemolysin in OMVs is a common feature among haemolytic strains. We propose an alternative pathway for the transport of the type I secreted alpha-haemolysin from the bacteria to the host cells during bacterial infections.