The present study demonstrates the important structural features of ceramide required for proper regulation, binding and identification by both pro-apoptotic and anti-apoptotic Bcl-2 family proteins. The C-4=C-5 trans-double bond has little influence on the ability of Bax and Bcl-xL to identify and bind to these channels. The stereochemistry of the headgroup and access to the amide group of ceramide is indispensible for Bax binding, indicating that Bax may interact with the polar portion of the ceramide channel facing the bulk phase. In contrast, Bcl-xL binding to ceramide channels is tolerant of stereochemical changes in the headgroup. The present study also revealed that Bcl-xL has an optimal interaction with long-chain ceramides that are elevated early in apoptosis, whereas short-chain ceramides are not well regulated. Inhibitors specific for the hydrophobic groove of Bcl-xL, including 2-methoxyantimycin A3, ABT-737 and ABT-263 provide insights into the region of Bcl-xL involved in binding to ceramide channels. Molecular docking simulations of the lowest-energy binding poses of ceramides and Bcl-xL inhibitors to Bcl-xL were consistent with the results of our functional studies and propose potential binding modes.

When activated, the proapoptotic protein Bax permeabilizes the mitochondrial outer membrane, allowing the release of proteins into the cytosol and thus initiating the execution phase of apoptosis. When activated Bax was reconstituted into phospholipid membranes, we discovered a new, to our knowledge, property of Bax channels: voltage gating. We also found that the same Bax sample under the same experimental conditions could give rise to two radically different channels: Type A, which is small, well behaved, homogeneous, and voltage-gated, and Type B, which is large, noisy, and voltage-independent. One Type B channel can be converted irreversibly into a population of Type A channels by the addition of La(3+). This conversion process appears to involve a two-dimensional budding mechanism. The existence of these two types of Bax channels suggests a process for controlling the degree of mitochondrial outer membrane permeabilization.

Bax, despite being a cytosolic protein, has the distinct ability to form channels in the mitochondrial outer membrane, which are capable of releasing proteins that initiate the execution phase of apoptosis. When studied in a planar phospholipid membrane system, full-length activated Bax can form conducting entities consistent with linearly organized three-channel units displaying steep voltage-gating (n=14) that rivals that of channels in excitable membranes. In addition, the channels display strong positive co-operativity possibly arising from the charge distribution of the voltage sensors. On the basis of functional behaviour, one of the channels in this functional triplet is oriented in the opposite direction to the others often resulting in conflicts between the effects of the electric field and the positive co-operativity of adjacent channels. The closure of the first channel occurs at positive potentials and this permits the second to close, but at negative potentials. The closure of the second channel in turn permits closure of the third, but at positive potentials. Positive co-operativity manifests itself in a number of ways including the second and the third channels opening virtually simultaneously. This extraordinary behaviour must have important, although as yet undefined, physiological roles.

This paper reports on the discovery of a novel three-membrane channel unit exhibiting very steep voltage dependence and strong cooperative behavior. It was reconstituted into planar phospholipid membranes formed by the monolayer method and studied under voltage-clamp conditions. The behavior of the novel channel-former, isolated from Escherichia coli, is consistent with a linearly organized three-channel unit displaying steep voltage-gating (a minimum of 14 charges in the voltage sensor) that rivals that of channels in mammalian excitable membranes. The channels also display strong cooperativity in that closure of the first channel permits the second to close and closure of the second channel permits closure of the third. All three have virtually the same conductance and selectivity, and yet the first and third close at positive potentials whereas the second closes at negative potentials. Thus, is it likely that the second channel-former is oriented in the membrane in a direction opposite to that of the other two. This novel structure is named “triplin.” The extraordinary behavior of triplin indicates that it must have important and as yet undefined physiological roles.

provide the first identification and characterization of the biophysical properties of the most prominently observed ion channel expressed within the inner and outer membrane of nuclei from adult skeletal muscle fibers. Excised insideout single channel recordings were obtained from individual nuclei acutely isolated from Flexor Digitorum Brevis (FDB) fibers of wild-type mice. The outer membrane of nuclei was readily accessible following isolation. For measurements of channels from the inner membrane, nuclei were treated with 1 % (w/v) sodium citrate in order to remove the outer membrane. We found that the predominant ion channel expressed in both the inner and outer nuclear membrane was a cationic channel that conducts monovalent ions with slight preference for potassium over sodium ions (a PK/PNa~1.22). A 10,000-fold difference in the concentration of free Ca2þ between the pipette and bath solutions did not affect the channel reversal potential in symmetric KCl (~0 mV), indicating that Ca2þ ions permeation is negligible. The maximum conductance of the channel in the outward direction was~162 pS. The mean open probability (PO) was~0.7 and voltage-independent between À50 mV to þ50 mV. We suggest that this novel monovalent cationic channel within the inner and outer membrane of skeletal muscle nuclei provides a countercurrent mechanism that minimizes voltage change across the nuclear membrane. This research was supported by NIH K01 award AR060831(to V.Y.) and NIH R01 grant AR44657 (to R.T.D).