Molecular template for a voltage sensor in a novel K+ channel. III. Functional reconstitution of a sensorless pore module from a prokaryotic Kv channel.

KvLm is a prokaryotic voltage-gated K(+) (Kv) channel from Listeria monocytogenes. The sequence of the voltage-sensing module (transmembrane segments S1-S4) of KvLm is atypical in that it contains only three of the eight conserved charged residues known to be deterministic for voltage sensing in eukaryotic Kv's. In contrast, the pore module (PM), including the S4-S5 linker and cytoplasmic tail (linker-S5-P-S6-C-terminus) of KvLm, is highly conserved. Here, the full-length (FL)-KvLm and the KvLm-PM only proteins were expressed, purified, and reconstituted into giant liposomes. The properties of the reconstituted FL-KvLm mirror well the characteristics of the heterologously expressed channel in Escherichia coli spheroplasts: a right-shifted voltage of activation, micromolar tetrabutylammonium-blocking affinity, and a single-channel conductance comparable to that of eukaryotic Kv's. Conversely, ionic currents through the PM recapitulate both the conductance and blocking properties of the FL-KvLm, yet the KvLm-PM exhibits only rudimentary voltage dependence. Given that the KvLm-PM displays many of the conduction properties of FL-KvLm and of other eukaryotic Kv's, including strict ion selectivity, we conclude that self-assembly of the PM subunits in lipid bilayers, in the absence of the voltage-sensing module, generates a conductive oligomer akin to that of the native KvLm, and that the structural independence of voltage sensing and PMs observed in eukaryotic Kv channels was initially implemented by nature in the design of prokaryotic Kv channels. Collectively, the results indicate that this robust functional module will prove valuable as a molecular template for coupling new sensors and to elucidate PM residue-specific contributions to Kv conduction properties.

Tim17p regulates the twin pore structure and voltage gating of the mitochondrial protein import complex TIM23.

The TIM23 complex mediates import of preproteins into mitochondria, but little is known of the mechanistic properties of this translocase. Here patch clamping reconstituted inner membranes allowed for first time insights into the structure and function of the preprotein translocase. Our findings indicate that the TIM23 channel has "twin pores" (two equal sized pores that cooperatively gate) thereby strikingly resembling TOM, the translocase of the outer membrane. Tim17p and Tim23p are homologues, but their functions differ. Tim23p acts as receptor for preproteins and may largely constitute the preprotein-conducting passageway. Conversely depletion of Tim17p induces a collapse of the twin pores into a single pore, whereas N terminus deletion or C terminus truncation results in variable sized pores that cooperatively gate. Further analysis of Tim17p mutants indicates that the N terminus is vital for both voltage sensing and protein sorting. These results suggest that although Tim23p is the main structural unit of the pore Tim17p is required for twin pore structure and provides the voltage gate for the TIM23 channel.

The mitochondrial channel VDAC has a cation-selective open state.

The mitochondrial channel VDAC is known to have two major classes of functional states, a large conductance "open" state that is anion selective, and lower conductance substates that are cation selective. The channel can reversibly switch between open and half-open states, with the latter predominant at increasing membrane voltages of either polarity. We report the presence of a new functional state of VDAC, a cation-selective state with conductance approximately equal to that of the canonical open state. This newly described state of VDAC can be reached from either the half-open cation-selective state or from the open anion-selective state. The latter transition implies that a mechanism exists for selectivity gating in VDAC that is separate from partial closure, which may be relevant to the physiological regulation of this channel and mitochondrial outer membrane permeability.

Electrophysiological approaches to the study of protein translocation in mitochondria.

Electrophysiological techniques have been integral to our understanding of protein translocation across various membranes, and, in particular, the mitochondrial inner and outer membranes. Descriptions of various methodologies (for example, patch clamp, planar bilayers, and tip dip, and their past and potential contributions) are detailed within. The activity of protein import channels of native mitochondrial inner and outer membranes can be studied by directly patch clamping mitochondria and mitoplasts (mitochondria stripped of their outer membrane by French pressing) from various genetically manipulated strains of yeast and mammalian tissue cultured cells. The channel activities of TOM, TIM23, and TIM22 complexes are compared with those reconstituted in proteoliposomes and with those of the recombinant proteins Tom40p, Tim23p, and Tim22p, which play major roles in protein translocation. Studies of the mechanism(s) and the role of channels in protein translocation in mitochondria are prototypes, as the same principles are likely followed in all biological membranes including the endoplasmic reticulum and chloroplasts. The ability to apply electrophysiological techniques to these channels is now allowing investigations into the role of mitochondria in diverse fields such as neurotransmitter release, long-term potentiation, and apoptosis.

Effects of cytochrome c on the mitochondrial apoptosis-induced channel MAC.

Recent studies indicate that cytochrome c is released early in apoptosis without loss of integrity of the mitochondrial outer membrane in some cell types. The high-conductance mitochondrial apoptosis-induced channel (MAC) forms in the outer membrane early in apoptosis of FL5.12 cells. Physiological (micromolar) levels of cytochrome c alter MAC activity, and these effects are referred to as types 1 and 2. Type 1 effects are consistent with a partitioning of cytochrome c into the pore of MAC and include a modest decrease in conductance that is dose and voltage dependent, reversible, and has an increase in noise. Type 2 effects may correspond to "plugging" of the pore or destabilization of the open state. Type 2 effects are a dose-dependent, voltage-independent, and irreversible decrease in conductance. MAC is a heterogeneous channel with variable conductance. Cytochrome c affects MAC in a pore size-dependent manner, with maximal effects of cytochrome c on MAC with conductance of 1.9-5.4 nS. The effects of cytochrome c, RNase A, and high salt on MAC indicate that size, rather than charge, is crucial. The effects of dextran molecules of various sizes indicate that the pore diameter of MAC is slightly larger than that of 17-kDa dextran, which should be sufficient to allow the passage of 12-kDa cytochrome c. These findings are consistent with the notion that MAC is the pore through which cytochrome c is released from mitochondria during apoptosis.

Control of mitochondrial protein import by pH.

Protein import into mitochondria is inhibited by protons (IC(50) pH 6.5). The channels of the import machinery were examined to further investigate this pH dependence. TOM and TIM23 are the protein translocation channels of the mitochondrial outer and inner membranes, respectively, and their single channel behaviors at various pHs were determined using patch-clamp techniques. While not identical, increasing H(+) concentration decreases the open probability of both TIM23 and TOM channels. The pattern of the pH dependences of protein import and channel properties suggests TIM23 open probability can limit import of nuclear-encoded proteins into the matrix of yeast mitochondria.