Aqueous channels are at the core of TOM and TIM23, the translocases mediating the transport of proteins across the outer and inner mitochondrial membranes. Yet, the existence of a channel associated to the TIM22 complex has been arguable, as its function relates to the insertion of multispanning proteins into the inner membrane. For the first time, we report conditions to detect a channel activity associated to the TIM22 translocase, in organello i.e. intact mitoplasts. An internal signal peptide in the intermembrane space of mitochondria is a requisite to induce this channel, otherwise silent. The channel shows slightly cationic and high conductance activity of 1000 pS with a predominant half-open substate. Despite their different composition, the channels of the three mitochondrial translocases are thus remarkably similar, in tenant with their common task as pores transiently clapping proteins in route to their final destination. Interestingly, low membrane potentials keep the channel fully open, providing a threshold level of the peptide is present. Our results portray TIM22 as a dynamic channel solely active in the presence of its cargo proteins. In its fully open conformation, favored by the combined action of internal signal peptide and low membrane potential, the channel could embrace the in transit protein. As insertion progresses, and initial interaction with signal peptide fades, the channel would close, sustaining its role as a shunt that places trapped proteins into the membrane.

Supported by grant 2PR04B005 from Junta de Extremadura. P.M.V. was a recipient of a CAPES fellowship (1047019).