We review data supporting a model in which activated tBID results in an allosteric activation of BAK, inducing its intramembranous oligomerization into a proposed pore for cytochrome c efflux. The BH3 domain of tBID is not required for targeting but remains on the mitochondrial surface where it is required to trigger BAK to release cytochrome c. tBID functions not as a pore-forming protein but as a membrane targeted and concentrated death ligand. tBID induces oligomerization of BAK, and both Bid and Bak knockout mice indicate the importance of this event in the release of cytochrome c. In parallel, the full pro-apoptotic member BAX, which is highly homologous to BAK, rapidly forms pores in liposomes that release intravesicular FITC-cytochrome c *20A Ê . A definable pore progressed from *11A Ê consisting of two BAX molecules to a *22A Ê pore comprised of four BAX molecules, which transported cytochrome c. Thus, an activation cascade of pro-apoptotic proteins from BID to BAK or BAX integrates the pathway from surface death receptors to the irreversible efflux of cytochrome c.

The BCL-2 family of proteins is composed of both pro-and antiapoptotic regulators, although its most critical biochemical functions remain uncertain. The structural similarity between the BCL-X L monomer and several ion-pore-forming bacterial toxins has prompted electrophysiologic studies. Both BAX and BCL-2 insert into KCl-loaded vesicles in a pH-dependent fashion and demonstrate macroscopic ion eff lux. Release is maximum at ϷpH 4.0 for both proteins; however, BAX demonstrates a broader pH range of activity. Both purified proteins also insert into planar lipid bilayers at pH 4.0. Single-channel recordings revealed a minimal channel conductance for BAX of 22 pS that evolved to channel currents with at least three subconductance levels. The final, apparently stable BAX channel had a conductance of 0.731 nS at pH 4.0 that changed to 0.329 nS when shifted to pH 7.0 but remained mildly Cl ؊ selective and predominantly open. When BAX-incorporated lipid vesicles were fused to planar lipid bilayers at pH 7.0, a Cl ؊ -selective (P K ͞P Cl ‫؍‬ 0.3) 1.5-nS channel displaying mild inward rectification was noted. In contrast, BCL-2 formed mildly K ؉ -selective (P K ͞P Cl ‫؍‬ 3.9) channels with a most prominent initial conductance of 80 pS that increased to 1.90 nS. Fusion of BCL-2-incorporated lipid vesicles into planar bilayers at pH 7.0 also revealed mild K ؉ selectivity (P K ͞P Cl ‫؍‬ 2.4) with a maximum conductance of 1.08 nS. BAX and BCL-2 each form channels in artificial membranes that have distinct characteristics including ion selectivity, conductance, voltage dependence, and rectification. Thus, one role of these molecules may include pore activity at selected membrane sites.

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Potassium channels have evolved to play specialized roles in both excitable and inexcitable tissues. Here we describe the cloning and expression of Slo3, a novel potassium channel abundantly expressed in mammalian spermatocytes. Slo3 represents a new and unique type of potassium channel regulated by both intracellular pH and membrane voltage. Reverse transcription-polymerase chain reaction, Northern analysis, and in situ hybridization show that Slo3 is primarily expressed in testis in both mouse and human. Because of its sensitivity to both pH and voltage, Slo3 could be involved in sperm capacitation and/or the acrosome reaction, essential steps in fertilization where changes in both intracellular pH and membrane potential are known to occur. The protein sequence of mSlo3 (the mouse Slo3 homologue) is similar to Slo1, the large conductance, calcium-and voltage-gated potassium channel. These results suggest that Slo channels comprise a multigene family, defined by a combination of sensitivity to voltage and a variety of intracellular factors. Northern analysis from human testis indicates that a Slo3 homologue is present in humans and conserved with regard to sequence, transcript size, and tissue distribution. Because of its high testisspecific expression, pharmacological agents that target human Slo3 channels may be useful in both the study of fertilization as well as in the control or enhancement of fertility.

A culture system of "giant" Drosophila neurons derived from cytokinesis-arrested embryonic neuroblasts was developed to overcome the technical difficulties usually encountered in studying small Drosophila neurons. Cytochalasin B-treated neuroblasts differentiated into giant multinucleated cells that displayed neuronal morphology and neuron-specific markers (Wu et al., 1990). Here, we report that these giant neurons express different excitability patterns and membrane channels similar to those reported in excitable tissues of Drosophila. Individual neurons exhibited distinct all-or-none or graded voltage responses upon current injection. Both current- and voltage-clamp recordings could be performed on the same neuron because of the large cell size, thus making it possible to elucidate the functional role of the individual types of channels. By using pharmacological agents and ion substitution, the following currents were identified in these giant neurons: inward Na+ and Ca2+ currents and outward voltage-activated (the A-type and delayed rectifier) and Ca(2+)-activated K+ currents. In addition, we found a tetrodotoxin (TTX)-sensitive, Na(+)-dependent outward K+ current and a persistent component of an inward Na+ current, which have not been reported in Drosophila previously. This culture system can be used to analyze the mutational perturbations in ion channels and the resultant alterations in membrane excitability. Neurons from the mutant slowpoke (slo), which is known to lack a component of the Ca(2+)-activated K+ currents in muscles, exhibited prolonged action potentials associated with defects in the Ca(2+)-activated K+ current. This abnormality appeared to be more severe in the neurites than in the soma.