Structural features of a multisubstate cardiac mitoplast anion channel: Inferences from single channel recording

Ion channels from sheep cardiac mitoplast (inverted inner mitochondrial membrane vesicle) preparations were incorporated into voltage-clamped planar lipid bilayers. A low-conductance anion channel (～40 or ～85 pS in symmetric 300 or 550 mM choline Cl, respectively), characterized by the presence of two well-defined substates, at ～25 and ～50% of the fully open level, was studied in detail. The substate behavior was consistent with a multibarrelled channel containing four functionally coupled pores. At negative (cis-trans) membrane potentials, the putative portomers appeared to gate with substantial positive cooperativity, accounting for the apparent absence of a ～75% sublevel. At positive holding potentials, allosteric protomer interactions were more complicated, and the channel complex could be modeled as a dimer of dimers. The protochannels in one dimer (“dimer A”) appeared to open independently of each other, and with a relatively high probability, while the monomers comprising the second dimer (“dimer B”) were functionally coupled, could only open if both protomers in dimer A were open, and closed as soon as one of the monomers in dimer A shut. The channels also displayed Ca²⁺- (and Mg²⁺-) sensitive rectification related to bilayer lipid surface charge. By assuming that Ca²⁺ acted solely by screening surface charge, the membrane surface potential profile was used as a “microscopic ruler” to place one mouth of the channel within 10–11 Å of the bilayer surface.