No abstract

Proteins of the Bcl-2 family are intracellular membrane-associated proteins that regulate programmed cell death (apoptosis) either positively or negatively by as yet unknown mechanisms. Bax, a pro-apoptotic member of the Bcl-2 family, was shown to form channels in lipid membranes. Bax triggered the release of liposome-encapsulated carboxyfluorescein at both neutral and acidic pH. At physiological pH, release could be blocked by Bcl-2. Bcl-2, in contrast, triggered carboxyfluorescein release at acidic pH only. In planar lipid bilayers, Bax formed pH- and voltage-dependent ion-conducting channels. Thus, the pro-apoptotic effects of Bax may be elicited through an intrinsic pore-forming activity that can be antagonized by Bcl-2.

The SS2 and adjacent regions of the 4 internal repeats of sodium channel II were subjected to single mutations involving, mainly, charged amino acid residues. These sodium channel mutants. expressed in Xerzupcts oocytes by microinjection of cDNA-derived mRNAs. were tested for sensitivity to tetrodotoxin and saxitoxin and for single-channel conductance.The resul!s obtained show that mutations involving 2 clusters of predominantly negatively charged residues. located at equivalent positions in the SS2 segment of the 4 repeats. strongly reduce toxin sensitivity. whereas mutations of adjacent residues exert much smaller or no effects. This suggests that the 2 clusters of residues. probably forming ring structures. take part in the extracellular mouth and/or the port wall of the sodium channel. This view is further supported by our Rnding that all mutations reducing net negative charge in these amino acid clusters cause a marked decrease in single-channel conductance.

Single-channel recordings of the currents mediated by the muscle Cl− channel, ClC-1, expressed in Xenopus oocytes, provide the first direct evidence that this channel has two equidistant open conductance levels like the Torpedo ClC-0 prototype. As for the case of ClC-0, the probabilities and dwell times of the closed and conducting states are consistent with the presence of two independently gated pathways with ≈ 1.2 pS conductance enabled in parallel via a common gate. However, the voltage dependence of the common gate is different and the kinetics are much faster than for ClC-0. Estimates of single-channel parameters from the analysis of macroscopic current fluctuations agree with those from single-channel recordings. Fluctuation analysis was used to characterize changes in the apparent double-gate behavior of the ClC-1 mutations I290M and I556N causing, respectively, a dominant and a recessive form of myotonia. We find that both mutations reduce about equally the open probability of single protopores and that mutation I290M yields a stronger reduction of the common gate open probability than mutation I556N. Our results suggest that the mammalian ClC-homologues have the same structure and mechanism proposed for the Torpedo channel ClC-0. Differential effects on the two gates that appear to modulate the activation of ClC-1 channels may be important determinants for the different patterns of inheritance of dominant and recessive ClC-1 mutations.

The oscillatory behavior of the cephalopod giant axons in response to an applied current has been established by previous investigators. In the study reported here the relationship between the familiar "RC" electrotonic response and the oscillatory behavior is examined experimentally and shown to be dependent on the membrane potential. Computations based on the threecurrent system which was inferred from electrical measurements by Hodgkin and Huxley yield subthreshold responses in good agreement with experimental data. The point which is developed explicitly is that since the three currents, in general, have nonzero resting values and two currents, the "Na" system and the "K" system, are controlled by voltage-dependent time-variant conductances, the subthreshold behavior of the squid axon in the small-signal range can be looked upon as arising from phenomenological inductance or capacitance. The total phenomenological impedance as a function of membrane potential is derived by linearizing the empirically fitted equations which describe the time-variant conductances. At the resting potential the impedance consists of three structures in parallel, namely, two series RL elements and one series RC element. The true membrane capacitance acts in parallel with the phenomenological elements, to give a total impedance which is, in effect, a parallel R, L, C system with a "natural frequency" of oscillation. At relatively hyperpolarized levels the impedance "degenerates" to an RC system. The subthreshold behavior of the axonal membrane, exemplified by the classical "electrotonus" of the myelinated nerve (Hermann, 1905; Hodgkin and Rushton, 1946;Davis and Lorente de N6, 1947) and the "local response" and oscillatory behavior of the giant axon in the cephalopods, Loligo and Sepia (Hodgkin, 1938;Arvanitaki, 1939 Arvanitaki, , 1941Brink et al., 1946), have been studied by many investigators. In recent years, perhaps due to the advent of the microelectrode technique with its emphasis on large amplitude recordings, electrophysiologists have shown only moderate interest in the subthreshold be-497