Electrostatic mutations in iberiotoxin as a unique tool for probing the electrostatic structure of the maxi-K channel outer vestibule.

Iberiotoxin (IbTX or alpha-KTx 1.3), a selective, high-affinity blocker of the large-conductance, calcium-activated (maxi-K) channel, exhibits a unique, asymmetric distribution of charge. To test how these charges control kinetics of IbTX binding, we generated five mutants at two positions, K27 and R34, that are highly conserved among other isotoxins. The dissociation and association rate constants, koff and kon, were determined from toxin-blocked and -unblocked durations of single maxi-K channels incorporated into planar lipid bilayers. Equilibrium dissociation constant (Kd) values were calculated from koff/kon. The IbTX mutants K27N, K27Q, and R34N caused large increases in Kd values compared to wild-type, suggesting that the IbTX interaction surface encompasses these residues. A well-established pore-blocking mechanism for IbTX predicts a voltage dependence of toxin-blocked times following occupancy of a potassium binding site in the channel pore. Time constants for block by K27R were approximately 5-fold slower at -20 mV versus +40 mV, while neutralization of K27 relieved the voltage dependence of block. This suggests that K27 in IbTX interacts with a potassium binding site in the pore. Neutralized mutants of K27 and R34, with zero net charge, displayed toxin association rate constants approximately 10-fold slower than wild-type. Association rates for R34N diminished approximately 19-fold when external potassium was increased from 30 to 300 mM. These findings suggest that simple net charge and diffusional processes do not control ingress of IbTX into the channel vestibule.

The beta subunit of the high-conductance calcium-activated potassium channel contributes to the high-affinity receptor for charybdotoxin.

Transient expression of either alpha or alpha + beta subunits of the high-conductance Ca2+-activated K+ (maxi-K) channel has been achieved in COS-1 cells. Expression has been studied using charybdotoxin (ChTX), a peptidyl inhibitor that binds in the pore on the alpha subunit. Although some properties of monoiodotyrosine-ChTX (125I-ChTX) binding to membranes derived from each type of transfected cells appear to be identical, other parameters of the binding reaction are markedly different. Under low ionic strength conditions, the affinity constant for 125I-ChTX measured under equilibrium binding conditions is increased ca. 50-fold in the presence of the beta subunit. The rate constant for 125I-ChTX association is enhanced ca. 5-fold, whereas the dissociation rate constant is decreased more than 7-fold when the beta subunit is present. These data indicate that functional coassembly of maxi-K channel subunits can be obtained in a transient expression system, and that the beta subunit has profound effects on 125I-ChTX binding. We postulate that certain negatively charged residues in the large extracellular loop of beta attract the positively charged 125I-ChTX to its binding site on alpha through electrostatic interactions, and account for effects observed on ligand association kinetics. Moreover, another residue(s) in the loop of beta must contribute to stabilization of the toxin-bound state, either by a direct interaction with toxin, or through an allosteric effect on the alpha subunit. Certain regions in the extracellular loop of the beta subunit may be in close proximity to the pore of the channel, and could play an important role in maxi-K channel function.

Brain pyruvate oxidation in experimental thiamin-deficiency encephalopathy.

Pyrithiamine-induced thiamin deficiency has been used in rat as an experimental form of Wernicke-Korsakoff encephalopathy, a disease associated with chronic alcoholism. Although the main etiological factor is known to be the lack of thiamin, the biochemical mechanisms involved in the pathogenesis remain unclear. Thiamin-dependent enzymes were studied in brain mitochondria: alpha-ketoglutarate dehydrogenase activity exhibited 40% reduction, whereas pyruvate dehydrogenase did not change significantly. Polarographic recordings of mitochondrial respiration revealed a decreased State 3, when using pyruvate/malate, alpha-ketoglutarate or glutamine as a substrate, but the respiration rates remained unchanged with glutamate or succinate. This fall in pyruvate oxidation may be due to the impairment of alpha-ketoglutarate dehydrogenase, which follows pyruvate dehydrogenase in the metabolic pathway. A time course of lactate concentration showed dramatic increases in thalamus, mid brain, hypothalamus and colliculli, consistent with the anatomopathological findings. No increases were found before the onset of neurological symptoms.

Characterization of tissue-expressed alpha subunits of the high conductance Ca(2+)-activated K+ channel.

Purified high conductance calcium-activated potassium (maxi-K) channels from tracheal smooth muscle have been shown to consist of a 60-70-kDa alpha subunit, encoded by the slo gene, and a 31-kDa beta subunit. Although the size of the beta subunit is that expected for the product of the gene encoding this protein, the size of the alpha subunit is smaller than that predicted from the slo coding region. To determine the basis for this discrepancy, sequence-directed antibodies have been raised against slo. These antibodies specifically precipitate the in vitro translation product of mslo, which yields an alpha subunit of the expected molecular mass (135 kDa). Immunostaining experiments employing smooth muscle sarcolemma, skeletal muscle T-tubules, as well as membranes derived from GH3 cells reveal the presence of an alpha subunit with an apparent molecular mass of 125 kDa. The difference in size of the alpha subunit as expressed in these membranes and the purified preparations is due to a highly reproducible proteolytic decay that occurs mostly at an advanced stage of the maxi-K channel purification. In the purified maxi-K channel preparations investigated, the full-length alpha subunit, an intermediate size product of 90 kDa, and the 65-kDa polypeptide, as well as other smaller fragments can be detected using appropriate antibodies. Proteolysis occurs exclusively at two distinct positions within the long C-terminal tail of slo. In addition, evidence for the tissue expression of distinct splice variants in membrane-bound as well as purified maxi-K channels is presented.

Cross-linking of charybdotoxin to high-conductance calcium-activated potassium channels: identification of the covalently modified toxin residue.

High-conductance calcium-activated potassium (maxi-K) channels are composed of two subunits, alpha and beta. The pore-forming alpha subunit is a member of the mSlo family of K+ channels, whereas the beta subunit is a novel protein that modulates the biophysical and pharmacological properties of the channel complex. In the presence of a bifunctional cross-linking reagent, monoiodotyrosine charybdotoxin ([125I]ChTX) is covalently incorporated specifically into Lys69 of the beta subunit, which is located in a large extracellular loop of this protein. Using variants of ChTX which retain their channel-blocking activity and in which individual Lys residues have been mutated, we have identified the corresponding amino acid in ChTX that is involved in the cross-linking reaction. All of the ChTX mutants investigated bind to the maxi-K channel and display the same pharmacological profile as native ChTX in competition binding experiments. Whereas substitution of amino acids at positions 11 and 31 of ChTX yields wild-type cross-linking patterns, the peptide without a Lys at position 32 fails to incorporate into the beta subunit of the maxi-K channel. Given the model for the interaction between ChTX and the outer vestibule of the maxi-K channel that has been proposed (Stampe et al., 1994), our data constrain the maximum distance between the pore of this channel and the region in the extracellular loop of the beta subunit where the cross-linking reaction takes place to 11 A. This topological limit helps define structural features of the maxi-K channel that may aide in probing the functional interaction between alpha and beta subunits of the channel complex.

Charybdotoxin and its effects on potassium channels.

Over the last few years, a considerable amount of information has been obtained regarding K+ channels. Different areas of research have contributed to knowledge in this field. Charybdotoxin (ChTX), a 37-amino acid peptide isolated from venom of the scorpion Leiurus quinquestriatus var. hebraeus, represents a remarkable tool for studying K+ channels. With its use, it has been possible to purify the high-conductance Ca(2+)-activated K+ (maxi-K) channel to homogeneity and determine the subunit composition of this channel. This has led to the discovery of an auxiliary beta-subunit that, when coexpressed with the pore-forming subunit, mSlo, alters the biophysical and pharmacological properties of this latter subunit. With the feasibility of producing large amounts of ChTX by recombinant techniques and the knowledge of the three-dimensional structure of the peptide, it has been possible to carry out site-directed mutagenesis studies and obtain a picture of the interaction surface of the toxin with two channels, maxi-K and Shaker, and to derive a picture of the complementary surface of the receptor in these two channels. Finally, ChTX, and the more selective K+ channel toxins that were subsequently discovered, have provided us with unique tools not only to determine the functional role that K+ channels play in target tissues but also to develop the molecular pharmacology of these channels.

Proto-oncogene c-fos induction in thiamine-deficient encephalopathy. Protective effects of nicardipine on pyrithiamine-induced lesions.

Treatment of rats with the central thiamine antagonist, pyrithiamine, results in severe neurological symptoms such as ataxia and convulsions. Induction of proto-oncogene c-fos expression, often related to seizure activity, has been detected in the brains of thiamine-deficient rats by means of Northern blot analysis and in situ hybridization. Region-selective increases of lactate observed following thiamine deficiency development are largely coincident with histologically vulnerable regions. When thiamine-deficient rats were treated with the calcium channel blocker, nicardipine, lesions associated with thiamine deficiency did not appear and there was no induction of c-fos mRNA expression. This suggests a neurocytoprotective role of nicardipine to neuronal cell damage in thiamine-deficient encephalopathy.

Assay of succinate dehydrogenase activity by a colorimetric-continuous method using iodonitrotetrazolium chloride as electron acceptor.

A spectrophotometric assay method for determining succinate dehydrogenase activity is described in which iodonitrotetrazolium chloride is used as a final electron acceptor. The enzyme activity is determined by measuring the formation of formazan due to the tetrazolium salt reduction. The assay is continuous, rapid, simple, and sensitive, and may be used in the determination of enzyme activity either in tissue homogenates or as a marker of the mitochondrial fraction in cell fractionation procedures.