The chemotherapeutic oxaliplatin alters voltage-gated Na(+) channel kinetics on rat sensory neurons.

The chemotherapeutic oxaliplatin causes a sensory-motor neuropathy with predominantly hyperpathic symptoms. The mechanism underlying this hyperexcitability was investigated using rat sensory nerve preparations, dorsal root ganglia and hippocampal neurons. Oxaliplatin resulted in an increase of the amplitude and duration of compound action potentials. It lengthened the refractory period of peripheral nerves suggesting an interaction with voltage-gated Na(+) channels. Application of oxaliplatin to dorsal root ganglion neurons resulted in an increase of the Na(+) current, a block of the maximal amplitude and a shift of the voltage-response relationship towards more negative membrane potentials. The effect was detectable on 13 of 18 tested cells. This observation, together with the absence of any effect on Na(+) currents of hippocampal neurons, suggests that the interaction of oxaliplatin is restricted to one or more channel subtypes. The effect of oxaliplatin could be antagonised by the Na(+) channel blocker carbamazepine which could be used to reduce side effects of oxaliplatin therapy in patients.

Activation of rat recombinant alpha(1)beta(2)gamma(2S) GABA(A) receptor by the insecticide ivermectin.

In the present study, the activation of rat recombinant alpha(1)beta(2)gamma(2S) gamma-aminobutyric acid (GABA)-ergic Cl(-) channel expressed in human embryonic kidney (HEK) 293 cells by ivermectin was investigated. Maximal activation of the channel occurred with GABA concentrations of 10 mM or 20 microM ivermectin both achieving about the same current amplitudes. With those saturating concentrations, the currents rose with GABA within 1 ms to the maximal values, whereas the rise time for ivermectin was about 500 times longer. In contrast to activation with GABA, no desensitisation in the presence of the agonist was observed with ivermectin. With both agonists, two different open states were detected. On simultaneous application of GABA and ivermectin the current amplitudes and the kinetics were determined by the agonist applied in the concentration eliciting the higher open probability. It is concluded that GABA and ivermectin activated the channel independently resulting in different kinetic properties.

Desensitization characteristics of rat recombinant GABA(A) receptors consisting of alpha1beta2gamma2S and alpha1beta2 subunits expressed in HEK293 cells.

Desensitization kinetics of rat recombinant typeA GABAergic receptors consisting of the subunits alpha1beta2gamma2S or alpha1beta2 was investigated on application of 10-0.001 mM GABA to whole cell patches using a piezo driven liquid filament switch for fast application and deapplication. At high GABA concentrations desensitization was triphasic showing increasing time constants and a decreasing extent of desensitization on lowering the GABA concentration. Below agonist concentrations of 1 mM for the trimeric receptor and 0.1 mM for the dimeric one desensitization was biphasic switching to monophasic kinetics at GABA concentrations < or = 0.01 mM for the alpha1beta2gamma2S-type and < or = 0.003 mM for the alpha1beta2-type, respectively. Comparison with former studies performed with GABAergic receptors consisting of different subunits revealed differences in the desensitization kinetics.

Molecular modulation of recombinant rat alpha1beta2gamma2 GABA(A) receptor channels by diazepam.

Recombinant gamma-aminobutyric acid (GABA(A)) receptor channels containing alpha1beta2gamma2-subunits were transiently expressed in HEK293 cells. Modulation by diazepam (DZ) was investigated using the patch-clamp technique with a device for ultra-fast solution exchange. GABA activated Cl(-)-currents were potentiated when DZ > 0.1 microM was added to non-saturating concentrations of GABA (< 0.1 mM GABA). Maximal potentiation of the peak current amplitude by a factor of 2.5 was observed when 1 microM DZ was added to the test-solution. Deactivation of GABA-activated currents after the end of GABA pulses was best fitted with two time constants. After application of DZ + GABA, increase of time constants of deactivation was measured. It was independent on GABA concentration. We conclude that prolongation of deactivation after application of GABA + DZ may be an important mechanism of the modulatory action of DZ at GABA(A) receptor channels.

The peripheral complex of the tobacco hornworm V-ATPase contains a novel 13-kDa subunit G.

A prominent 16-kDa protein copurifies with the V-ATPase isolated from both posterior midgut and Malpighian tubules of Manduca sexta larvae and thus was believed to represent a V-ATPase subunit. [14C]N,N'-dicyclohexylcarbodiimide labeling and its position on SDS-electrophoresis gels revealed that this protein was different from the 17-kDa proteolipid. A cDNA clone encoding a highly hydrophilic protein with a calculated molecular mass of 13,692 Da was obtained by immunoscreening. Monospecific antibodies, affinity-purified to the 13-kDa recombinant protein expressed in Escherichia coli, specifically recognized the 16-kDa protein of the purified V-ATPase, confirming that a cDNA encoding this protein had been cloned. In vitro translation of the cRNA showed that the cloned 13-kDa subunit behaved like a 16-kDa protein on SDS-electrophoresis gels. The cloned protein showed 37% amino acid sequence identity to the 13-kDa V-ATPase subunit Vma10p recently cloned from yeast and some similarity to subunit b of bacterial F-ATPases. In contrast to the Vma10p protein, which behaved like a V0 subunit, the M. sexta 13-kDa protein behaved like a V1 subunit, since it could be stripped from the membrane by treatment with the chaotropic salt KI and by cold inactivation. When KI dissociated V-ATPase subunits were reassociated by dialysis that removed the KI, a soluble, 450-kDa complex of the M. sexta V-ATPase could be purified by gel chromatography. This V1 complex consisted of subunits A, B, E, and the 13-kDa subunit, confirming that the cloned protein is a new V-ATPase subunit and a member of the peripheral V1 complex of the V-ATPase. We designate this new V1 component subunit G.

K+/H+ antiport in the tobacco hornworm midgut: the K(+)-transporting component of the K+ pump.

The midgut of the tobacco hornworm secretes K+ across the apical plasma membrane of its goblet cells. This secondary K+ transport results from K+/H+ antiport energized by the proton-motive force generated by a primary, H(+)-transporting plasma membrane V-ATPase. Thus, the lepidopteran midgut constitutes a well-established example of the emerging concept that the proton-motive force is an alternative to the classical sodium-motive force for the energization of animal plasma membranes. K+/H+ antiport in the tobacco hornworm midgut is electrophoretic, exchanging 2H+ for 1K+. Under physiological conditions, it is energized by the voltage component of the proton-motive force. The strong coupling of electrophoretic K+/2H+ antiport with the electrogenic V-ATPase provides, in principle, the minimal device for the alkalization of the midgut lumen to pH values higher than 11. K+/H+ antiport is insensitive to bafilomycin A1, but is inhibited by amiloride or Concanavalin A. Lectin staining of blots after SDS-PAGE revealed several glycosylated polypeptides in the goblet cell apical membrane which are not part of the V-ATPase and thus are candidates for the antiporter protein. Current efforts are focused on the isolation of the K+/H+ antiporter.

A novel 14-kDa V-ATPase subunit in the tobacco hornworm midgut.

A cDNA clone encoding a hydrophilic protein with a calculated molecular mass of 13,839 Da was isolated by shotgun screening with an anti-V-ATPase holoenzyme serum. The deduced amino acid sequence showed no significant homology to any other known protein. Southern blots revealed the existence of only one gene encoding the 14-kDa protein. Monospecific antibodies purified by affinity to the recombinant protein demonstrated the presence of a 14-kDa protein in the highly purified goblet cell apical membrane and inhibited ATP-dependent proton transport as well as V-ATPase activity to the same extent. Thus, the 14-kDa protein was shown to be a part of the V-ATPase holoenzyme. Binding of the monospecific antibodies to the ATPase seemed to require an ATP-dependent conformational change of the enzyme, since inhibition only occurred when ATP was present during the antibody binding step. The 14-kDa subunit could be stripped from the membrane by treatment with the chaotropic agent KI, confirming it to be part of the soluble complex of the V-ATPase. In immunoblots, the 14-kDa-specific antibodies showed no cross-reaction with several xenic V-ATPases.

Specific recognition of the 3'-terminal adenosine of tRNAPhe in the exit site of Escherichia coli ribosomes.

Ribosomes from Escherichia coli possess, in addition to A and P sites, a third tRNA binding site, which according to its presumed function in tRNA release during translocation has been termed the exit site. The exit site exhibits a remarkable specificity for deacylated tRNA; charged tRNA, e.g. N-AcPhe-tRNAPhe, is not bound significantly. To determine the molecular basis of this discrimination, we have measured the exit site binding affinities of a number of derivatives of tRNAPhe from E. coli, modified at the 3' end. Binding to the exit site of the tRNAPhe derivatives was measured fluorimetrically by competition with a fluorescent tRNAPhe derivative. We show here that removal of the 2' and 3' hydroxyl groups of the 3'-terminal adenosine decreases the affinity of tRNAPhe for the exit site 15 and 40-fold, respectively. Substitutions at the 3' hydroxyl group (aminoacylation, phosphorylation, cytidylation) as well as removal of the 3'-terminal adenosine (or adenylate) of tRNAPhe lower the affinity below the detection limit of 2 x 10(5) M-1, i.e. more than 100-fold. Modification of the adenine moiety (1,N6-etheno adenine) or replacement of it with other bases (cytosine, guanine) has the same dramatic effect. In contrast, the binding to both P and A sites is virtually unaffected by all of the modifications tested. These results suggest that a major fraction (at least -12 kJ/mol, probably about -17 kJ/mol) of the free energy of exit site binding of tRNAPhe (-42 kJ/mol at 20 mM-Mg2+) is contributed by the binding of the 3'-terminal adenine to the ribosome. The binding most likely entails the formation of hydrogen bonds.