No abstract

SUMMARY1. The bee venom peptide, apamin, has been radiolabelled with 1251, the monoiodinated derivative purified, and its binding to intact guinea-pig liver cells studied.2. At 37 IC 1251-monoiodoapamin associated with, and dissociated from, guinea-pig hepatocytes remarkably rapidly. The association and dissociation rate constants were 1-4 x 108 M-1 s51 and 0 035 s51 respectively.3. Equilibrium binding studies demonstrated a saturable binding component compatible with 1:1 binding to a single class of site and having an equilibrium dissociation constant (KL) of 390 pm. The maximal binding capacity was 1 1 fmol mg-1 dry wt. of tissue. Unlabelled apamin displaced bound 1251-monoiodoapamin with a K, of 380 pm, which is consistent with the concentration of apamin required to inhibit Ca2+-activated K+ permeability (WK(ca)) in these cells.4. Inhibitable binding of '251-monoiodoapamin to rat hepatocytes was much less than to guinea-pig hepatocytes and could not be reliably quantified. Neither was there any discernible inhibitable binding to human erythrocytes. This is in keeping with the reported lack of apamin-sensitive Ca2+-activated K+ channels in these cell types.5. Various agents were tested for their ability to inhibit monoiodoapamin binding to, and Ca2+-mediated K+ efflux from, guinea-pig hepatocytes. All compounds tested which inhibited binding also blocked K+ efflux at similar concentrations. TEA and quinine affected hepatocytes only at high concentration (KI = 5-8 and 0'51 mM respectively). 9-aminoacridine, quinacrine and chloroquine were slightly more effective (KI = 70-180 ItM). By far the most active compounds (apart from apamin) were the neuromuscular blocking agents; tubocurarine, pancuronium and atracurium (KI = 7-5, 6'8 and 4'5 M respectively). Gallamine was slightly less effective (KI = 14 /LM) and decamethonium and hexamethonium much less so (KI = 620 and 760 ,uM respectively).6. 3,4-diaminopyridine, a-bungarotoxin and tetrodotoxin were among several compounds which showed little or no affinity for apamin binding sites or inhibition of K+ efflux in guinea-pig hepatocytes.

The advantages of using isolated cells have led us to develop short-term cultures of hippocampal pyramidal cells, which retain many of the properties of cells in acute preparations and in particular the ability to generate afterhyperpolarizations after a train of action potentials. Using perforated-patch recordings, both medium and slow afterhyperpolarization currents (mI(AHP) and sI(AHP), respectively) could be obtained from pyramidal cells that were cultured for 8-15 days. The sI(AHP) demonstrated the kinetics and pharmacologic characteristics reported for pyramidal cells in slices. In addition to confirming the insensitivity to 100 nM apamin and 1 mM TEA, we have shown that the sI(AHP) is also insensitive to 100 nM charybdotoxin but is inhibited by 100 microM D-tubocurarine. Concentrations of nifedipine (10 microM) and nimodipine (3 microM) that maximally inhibit L-type calcium channels reduced the sI(AHP) by 30 and 50%, respectively. However, higher concentrations of nimodipine (10 microM) abolished the sI(AHP), which can be partially explained by an effect on action potentials. Both nifedipine and nimodipine at maximal concentrations were found to reduce the HVA calcium current in freshly dissociated neurons to the same extent. The N-type calcium channel inhibitor, omega-conotoxin GVIA (100 nM), irreversibly inhibited the sI(AHP) by 37%. Together, omega-conotoxin (100 nM) and nifedipine (10 microM) inhibited the sI(AHP) by 70%. 10 microM ryanodine also reduced the sI(AHP) by 30%, suggesting a role for calcium-induced calcium release. It is concluded that activation of the sI(AHP) in cultured hippocampal pyramidal cells is mediated by a rise in intracellular calcium involving multiple pathways and not just entry via L-type calcium channels.

The rat SK1 gene (rSK1) does not form functional Ca2+‐activated potassium channels when expressed alone in mammalian cell lines. Using a selective antibody to the rSK1 subunit and a yellow fluorescent protein (YFP) tag we have discovered that rSK1 expression produces protein that remains largely at intracellular locations. We tested the idea that rSK1 may need an expression partner, rSK2, in order to form functional channels. When rSK1 was co‐expressed with rSK2 in HEK 293 cells it increased the current magnitude by 77 ± 34 % (as compared with cells expressing rSK2 alone). Co‐expression of rSK1 with rSK2 also changed the channel pharmacology. The sensitivity of SK current to block by apamin was reduced ~16‐fold from an IC50 of 94 pm (for SK2 alone) to 1.4 nm (for SK2 and SK1 together). The sensitivity to block by UCL 1848 (a potent small molecule blocker of SK channels) was similarly reduced, ~26‐fold, from an IC50 of 110 pm to 2.9 nm. These data clearly demonstrate that rSK1 and rSK2 subunits interact. The most likely explanation for this is that the subunits are able to form heteromeric assemblies.

The pharmacology of hSK1, a small conductance calcium-activated potassium channel, was studied in mammalian cell lines (HEK293 and COS-7). In these cell types, hSK1 forms an apamin-sensitive channel with an IC 50 for apamin of 8 nM in HEK293 cells and 12 nM in COS-7 cells. The currents in HEK293 cells were also sensitive to tubocurarine (IC 50 =23 mM), dequalinium (IC 50 =0.4 mM), and the novel dequalinium analogue, UCL1848 (IC 50 =1 nM). These results are very dierent from the pharmacology of hSK1 channels expressed in Xenopus oocytes and suggest the properties of the channel may depend on the expression system. Our ®ndings also raise questions about the role of SK1 channels in generating the apamin-insensitive slow afterhyperpolarization observed in central neurones.