The biophysical properties of small conductance Ca(2+)-activated K(+) (SK) channels are well suited to underlie afterhyperpolarizations (AHPs) shaping the firing patterns of a conspicuous number of central and peripheral neurons. We have identified a new scorpion toxin (tamapin) that binds to SK channels with high affinity and inhibits SK channel-mediated currents in pyramidal neurons of the hippocampus as well as in cell lines expressing distinct SK channel subunits. This toxin distinguished between the SK channels underlying the apamin-sensitive I(AHP) and the Ca(2+)-activated K(+) channels mediating the slow I(AHP) (sI(AHP)) in hippocampal neurons. Compared with related scorpion toxins, tamapin displayed a unique, remarkable selectivity for SK2 versus SK1 ( approximately 1750-fold) and SK3 ( approximately 70-fold) channels and is the most potent SK2 channel blocker characterized so far (IC(50) for SK2 channels = 24 pm). Tamapin will facilitate the characterization of the subunit composition of native SK channels and help determine their involvement in electrical and biochemical signaling.

Taicatoxin, isolated from the venom of the Australian taipan snake Oxyuranus scutellatus, has been previously regarded as a specific blocker of high threshold Ca2+ channels in heart. Here we show that taicatoxin (in contrast to a range of other Ca2+ channel blockers) interacts with apamin-sensitive, small conductance, Ca2+-activated potassium channels on both chromaffin cells and in the brain. Taicatoxin displays high affinity recognition of125I-apamin acceptor-binding sites, present on rat synaptosomal membranes (K i = 1.45 ± 0.22 nm) and also specifically blocks affinity-labeling of a 33-kDa 125I-apamin-binding polypeptide on rat brain membranes. Taicatoxin (50 nm) completely blocks apamin-sensitive after-hyperpolarizing slow tail K+currents generated in rat chromaffin cells (mean block 97 ± 3%,n = 12) while only partially reducing total voltage-dependent Ca2+ currents (mean block 12 ± 4%, n = 6). In view of these findings, the use of taicatoxin as a specific ligand for Ca2+ channels should now be reconsidered.

I-125-Apamin and photolabile derivatives of the toxin have been used to investigate the binding properties and subunit composition of small conductance Ca2+-activated potassium channels (SKCa, channels) expressed on plasma membranes from rat brain, rabbit liver, or rat pheochromocytoma (PC12) cells, On all preparations, I-125-apamin recognized single classes of acceptor binding sites with similar high affinity (Kd similar to 3-6 pM), Gallamine, however, was found to readily discriminate between I-125-apamin accepters present in these preparations, showing a maximal approx ninefold difference in affinity for accepters expressed by rabbit liver or PC 12 cells. Affinity-labeling patterns revealed the expression of different hetero-oligomeric combinations of high (86 or 59 kDa) and low (33 or 30 kDa) molecular mass I-125-apamin-binding polypeptides, consistent with pharmacological differences. Alternative expression of either 86- or 59-kDa polypeptides appeared to be the most important factor influencing gallamine's affinity for SKCa channel subtypes, Both high- and low-molecular-mass polypeptides are integral membrane proteins, the latter being glycosylated in a tissue-specific manner. (C) 1997 Academic Press.

Small-conductance calcium-activated potassium channels (SK channels) can be operationally defined as those having single-channel conductance of less than 20 pS. These channels are found in a wide range of excitable and non-excitable cells. In nerve and muscle, the ionic currents that flow through these channels are responsible for maintaining the slow after-hyperpolarizing potential (AHP) that follows bursts of action potentials. Two venom toxins have been characterized that appear to interact specifically with SK channels: apamin and leiurotoxin. Apamin, a neurotoxin isolated from the venom of the European honey bee Apis mellifera, is the first potassium channel toxin to be isolated and characterized. It is a basic peptide with a molecular weight of 2000. It has been used as a pharmacological tool to characterize ionic currents flowing through SK channels and for the quantification of apamin-binding sites and the identification of putative SK channel polypeptides. Apamin-binding assays are used to monitor the solubilization and purification of SK channel proteins. The toxin is also used to provide evidence of a regulatory role for SK channels in cell metabolism.

Apamin, a peptide neurotoxin from bee venom, blocks small conductance Ca(2+)-activated K+ channels in central synapses and peripheral tissues. Using 125I-apamin, single classes of high affinity binding sites (Kd 1-3 pM) were identified on plasma membranes from rat, rabbit, guinea pig, and bovine brain and from rabbit, guinea pig, and bovine liver. Binding was sensitive to scyllatoxin, dequalinium, gallamine, and d-tubocurarine but not to charybdotoxin, toxin I, or mast cell degranulating peptide. In contrast, saturable binding of 125I-apamin to rat liver plasma membranes was virtually undetectable, thereby providing a correlation with the ability to measure apamin-sensitive Ca(2+)-activated potassium currents in rabbit and guinea pig hepatocytes but not in rat hepatocytes. In agreement with membrane binding studies, homobifunctional cross-linkers identified apparently identical 33-kDa 125I-apamin binding polypeptides on brain plasma membranes from all species and analogous but distinct polypeptides on plasma membranes from rabbit, guinea pig, and bovine liver. None of these affinity-labeled polypeptides were detectable on plasma membranes from rat liver. Affinity labeling was abolished on both liver and brain membranes by apamin, scyllatoxin, dequalinium, gallamine, and d-tubocurarine. These results indicate that comparable approximately 30-kDa polypeptides may fulfill equivalent functional roles within putative subtypes of apamin-sensitive small conductance Ca(2+)-activated K+ channels.