Bax, but not Bcl-xL, decreases the lifetime of planar phospholipid bilayer membranes at subnanomolar concentrations.
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Release of proteins through the outer mitochondrial membrane can be a critical step in apoptosis, and the localization of apoptosis-regulating Bcl-2 family members there suggests they control this process. We used planar phospholipid membranes to test the effect of full-length Bax and Bcl-xL synthesized in vitro and native Bax purified from bovine thymocytes. Instead of forming pores with reproducible conductance levels expected for ionic channels, Bax, but not Bcl-xL, created arbitrary and continuously variable changes in membrane permeability and decreased the stability of the membrane, regardless of whether the source of the protein was synthetic or native. This breakdown of the membrane permeability barrier and destabilization of the bilayer was quantified by using membrane lifetime measurements. Bax decreased membrane lifetime in a voltage- and concentration-dependent manner. Bcl-xL did not protect against Bax-induced membrane destabilization, supporting the idea that these two proteins function independently. Corresponding to a physical theory for lipidic pore formation, Bax potently diminished the linear tension of the membrane (i.e., the energy required to form the edge of a new pore). We suggest that Bax acts directly by destabilizing the lipid bilayer structure of the outer mitochondrial membrane, promoting the formation of a pore-the apoptotic pore-large enough to allow mitochondrial proteins such as cytochrome c to be released into the cytosol. Bax could then enter and permeabilize the inner mitochondrial membrane through the same hole. (+info)
Differential modulation by copper and zinc of P2X2 and P2X4 receptor function.
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Differential Modulation by Copper and Zinc of P2X2 and P2X4 Receptor Function. The modulation by Cu2+ and Zn2+ of P2X2 and P2X4 receptors expressed in Xenopus oocytes was studied with the two-electrode, voltage-clamp technique. In oocytes expressing P2X2 receptors, both Cu2+ and Zn2+, in the concentration range 1-130 microM, reversibly potentiated current activated by submaximal concentrations of ATP. The Cu2+ and Zn2+ concentrations that produced 50% of maximal potentiation (EC50) of current activated by 50 microM ATP were 16.3 +/- 0.9 (SE) microM and 19.6 +/- 1.5 microM, respectively. Cu2+ and Zn2+ potentiation of ATP-activated current was independent of membrane potential between -80 and +20 mV and did not involve a shift in the reversal potential of the current. Like Zn2+, Cu2+ increased the apparent affinity of the receptor for ATP, as evidenced by a parallel shift of the ATP concentration-response curve to the left. However, Cu2+ did not enhance ATP-activated current in the presence of a maximally effective concentration of Zn2+, suggesting a common site or mechanism of action of Cu2+ and Zn2+ on P2X2 receptors. For the P2X4 receptor, Zn2+, from 0.5 to 20 microM enhanced current activated by 5 microM ATP with an EC50 value of 2.4 +/- 0.2 microM. Zn2+ shifted the ATP concentration-response curve to the left in a parallel manner, and potentiation by Zn2+ was voltage independent. By contrast, Cu2+ in a similar concentration range did not affect ATP-activated current in oocytes expressing P2X4 receptors, and Cu2+ did not alter the potentiation of ATP-activated current produced by Zn2+. The results suggest that Cu2+ and Zn2+ differentially modulate the function of P2X2 and P2X4 receptors, perhaps because of differences in a shared site of action on both subunits or the absence of a site for Cu2+ action on the P2X4 receptor. (+info)
Enhancement of bistability in spinal motoneurons in vivo by the noradrenergic alpha1 agonist methoxamine.
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Enhancement of bistability in spinal motoneurons in vivo by the noradrenergic alpha1 agonist methoxamine. Like many types of motoneurons, spinal motoneurons in the adult mammal can exhibit bistable behavior. This means that short periods of excitatory input can initiate long periods of self-sustained firing and that equally short periods of inhibition can return the cell to the quiescent state. Usually, the presence of one of the monoamines (either serotonin or norepinephrine) is required for spinal motoneurons to express bistable behaviors. Because the decerebrate cat preparation has tonic activity in monoaminergic fibers that originate in the brain stem and project to spinal motoneurons, these cells sometimes exhibit bistable behavior. However, exogenous application of the noradrenergic alpha1 agonist methoxamine greatly enhances bistable behavior in the decerebrate. The goal of this study was to identify the mechanisms of this action of methoxamine. The total persistent inward current (IPIC) in spinal motoneurons in the decerebrate cat was measured from I-V functions generated by triangular voltage commands applied using discontinuous single electrode voltage clamp. The effect of methoxamine on IPIC was assessed by comparing its properties in a control cell sample without methoxamine to its properties in a sample of cells obtained after application of methoxamine. In most experiments, at least one cell was obtained from each sample. Our results showed that methoxamine approximately doubled the amplitude of IPIC without changing its onset voltage, its offset voltage, or its persistence. The reduced amplitude was a consistent finding within experiments and so was unlikely to be caused by interanimal variability. In addition, methoxamine depolarized motoneurons without altering their input conductances, so that a smaller amount of current was required to reach the onset voltage of IPIC. These effects of methoxamine were approximately equal in all cells. As a result of these changes, methoxamine greatly enhanced the tendency for motoneurons to become bistable. It is proposed that the methoxamine-induced increase in the amplitude of IPIC is effective in enhancing the duration of bistable firing because this increase makes IPIC more resistant to the deactivating effects of the afterhyperpolarizations between spikes. (+info)
Contribution of potassium conductances to a time-dependent transition in electrical properties of a cockroach motoneuron soma.
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Contribution of potassium conductances to a time-dependent transition in electrical properties of a cockroach motoneuron soma. The cell body of the cockroach (Periplaneta americana) fast coxal depressor motoneuron (Df) displays a time-dependent change in excitability. Immediately after dissection, depolarization evokes plateau potentials, but after several hours all-or-none action potentials are evoked. Because K channel blockers have been shown to produce a similar transition in electrical properties, we have used current-clamp, voltage-clamp and action-potential-clamp recording to elucidate the contribution of different classes of K channel to the transition in electrical activity of the neuron. Apamin had no detectable effect on the neuron, but charybdotoxin (ChTX) caused a rapid transition from plateau potentials to spikes in the somatic response of Df to depolarization. In neurons that already produced spikes when depolarized, ChTX increased spike amplitude but did not increase their duration nor decrease the amplitude of their afterhyperpolarization. 4-Aminopyridine (4-AP) (which selectively blocks transient K currents) did not cause a transition from plateau potentials to spikes but did enhance oscillations superimposed on plateau potentials. When applied to neurons that already generated spikes when depolarized, 4-AP could augment spike amplitude, decrease the latency to the first spike, and prolong the afterhyperpolarization. Evidence suggests that the time-dependent transition in electrical properties of this motoneuron soma may result, at least in part, from a fall in calcium-dependent potassium current (IK,Ca), consequent on a gradual reduction in [Ca2+ ]i. Voltage-clamp experiments demonstrated directly that outward K currents in this neuron do fall with a time course that could be significant in the transition of electrical properties. Voltage-clamp experiments also confirmed the ineffectiveness of apamin and showed that ChTX blocked most of IK,Ca. Application of Cd2+ (0.5 mM), however, caused a small additional suppression in outward current. Calcium-insensitive outward currents could be divided into transient (4-AP-sensitive) and sustained components. The action-potential-clamp technique revealed that the ChTX-sensitive current underwent sufficient activation during the depolarizing phase of plateau potentials to enable it to shunt inward conductances. Although the ChTX-sensitive conductance apparently makes little contribution to spike repolarization, the ChTX-resistant IK,Ca does make a significant contribution to this phase of the action potential. The 4-AP-sensitive current began to develop during the rising phase of both action potentials and plateau potentials but had little effect on the electrical activity of the neuron, probably because of its relatively small amplitude. (+info)
BK channel activation by brief depolarizations requires Ca2+ influx through L- and Q-type Ca2+ channels in rat chromaffin cells.
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BK channel activation by brief depolarizations requires Ca2+ influx through L- and Q-type Ca2+ channels in rat chromaffin cells. Ca2+- and voltage-dependent BK-type K+ channels contribute to action potential repolarization in rat adrenal chromaffin cells. Here we characterize the Ca2+ currents expressed in these cells and identify the Ca2+ channel subtypes that gate the activation of BK channels during Ca2+ influx. Selective Ca2+ channel antagonists indicate the presence of at least four types of high-voltage-gated Ca2+ channels: L-, N-, P, and Q type. Mean amplitudes of the L-, N-, P-, and Q-type Ca2+ currents were 33, 21, 12, and 24% of the total Ca2+ current, respectively. Five-millisecond Ca2+ influx steps to 0 mV were employed to assay the contribution of Ca2+ influx through these Ca2+ channels to the activation of BK current. Blockade of L-type Ca2+ channels by 5 microM nifedipine or Q-type Ca2+ channels by 2 microM Aga IVA reduced BK current activation by 77 and 42%, respectively. In contrast, blockade of N-type Ca2+ channels by brief applications of 1-2 microM CnTC MVIIC or P-type Ca2+ channels by 50-100 nM Aga IVA reduced BK current activation by only 11 and 12%, respectively. Selective blockade of L- and Q-type Ca2+ channels also eliminated activation of BK current during action potentials, whereas almost no effects were seen by the selective blockade of N- or P-type Ca2+ channels. Finally, the L-type Ca2+ channel agonist Bay K 8644 promoted activation of BK current by brief Ca2+ influx steps by more than twofold. These data show that, despite the presence of at least four types of Ca2+ channels in rat chromaffin cells, BK channel activation in rat chromaffin cells is predominantly coupled to Ca2+ influx through L- and Q-type Ca2+ channels. (+info)
Block of quantal end-plate currents of mouse muscle by physostigmine and procaine.
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of quantal end-plate currents of mouse muscle by physostigmine and procaine. Quantal endplate currents (qEPCs) were recorded from hemidiaphragms of mice by means of a macro-patch-clamp electrode. Excitation was blocked with tetrodotoxin, and quantal release was elicited by depolarizing pulses through the electrode. Physostigmine (Phys) or procaine (Proc) was applied to the recording site by perfusion of the electrode tip. Low concentrations of Phys increased the amplitude and prolonged the decay time constants of qEPCs from approximately 3 to approximately 10 ms, due to block of acetylcholine-esterase. With 20 microM to 2 mM Phys or Proc, the decay of qEPCs became biphasic, an initial short time constant taus decreasing to <1 ms with 1 mM Phys and to approximately 0.3 ms with 1 mM Proc. The long second time constant of the decay, taul, reached values of +info)
Physiological properties of GABAA receptors from acutely dissociated rat dentate granule cells.
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Physiological properties of GABAA receptors from acutely dissociated rat dentate granule cells. Study of fast, GABAA receptor-mediated, inhibitory postsynaptic currents (IPSCs) in hippocampal dentate granule cells has suggested that properties of GABAA receptors influence the amplitude and time course of the IPSCs. This study describes the physiological properties of GABAA receptors present on hippocampal dentate granule cells acutely isolated from 18- to 35-day-old rats. Rapid application of 1 mM GABA to outside-out macropatches excised from granule cells produced GABAA receptor currents with rapid rise time and biexponential decay of current after removal of GABA. After activation, granule cell GABAA receptor currents desensitized incompletely. During a 400-ms application of 1 mM GABA, peak current only desensitized approximately 40%. In symmetrical chloride solutions there was no outward rectification of whole cell current. Activation rates and peak currents elicited by rapid application of GABA to macropatches were also similar at positive and negative holding potentials. However, deactivation of GABAA receptor currents was slower at positive holding potentials. When whole cell currents were recorded without ATP in the pipette, current run-down was not apparent for 30 min in 50% of neurons, but run-down appeared to start soon after access was established in the remaining neurons. When 2 mM ATP was included in the recording pipette no run-down was apparent in 30 min of recording. The efficacy and potency of GABA were lower in cells recorded with no ATP in the pipette and during run-down compared with those recorded with 2 mM ATP and no run-down. (+info)
Auxiliary Hyperkinetic beta subunit of K+ channels: regulation of firing properties and K+ currents in Drosophila neurons.
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Auxiliary Hyperkinetic beta subunit of K+ channels: regulation of firing properties and K+ currents in Drosophila neurons. Molecular analysis and heterologous expression have shown that K+ channel beta subunits regulate the properties of the pore-forming alpha subunits, although how they influence neuronal K+ currents and excitability remains to be explored. We studied cultured Drosophila "giant" neurons derived from mutants of the Hyperkinetic (Hk) gene, which codes for a K+ channel beta subunit. Whole cell patch-clamp recording revealed broadened action potentials and, more strikingly, persistent rhythmic spontaneous activities in a portion of mutant neurons. Voltage-clamp analysis demonstrated extensive alterations in the kinetics and voltage dependence of K+ current activation and inactivation, especially at subthreshold membrane potentials, suggesting a role in regulating the quiescent state of neurons that are capable of tonic firing. Altered sensitivity of Hk currents to classical K+ channel blockers (4-aminopyridine, alpha-dendrotoxin, and TEA) indicated that Hk mutations modify interactions between voltage-activated K+ channels and these pharmacological probes, apparently by changing both the intra- and extracellular regions of the channel pore. Correlation of voltage- and current-clamp data from the same cells indicated that Hk mutations affect not only the persistently active neurons, but also other neuronal categories. Shaker (Sh) mutations, which alter K+ channel alpha subunits, increased neuronal excitability but did not cause the robust spontaneous activity characteristic of some Hk neurons. Significantly, Hk Sh double mutants were indistinguishable from Sh single mutants, implying that the rhythmic Hk firing pattern is conferred by intact Shalpha subunits in a distinct neuronal subpopulation. Our results suggest that alterations in beta subunit regulation, rather than elimination or addition of alpha subunits, may cause striking modifications in the excitability state of neurons, which may be important for complex neuronal function and plasticity. (+info)