Paxilline Inhibition of the Alpha-subunit of the High-conductance Calcium-activated Potassium Channel

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Abstract

High conductance calcium-activated (maxi-K) channels are potently blocked by a family of indole diterpenes that includes paxilline. Paxilline stimulates binding of charybdotoxin (ChTX) to maxi-K channels in vascular smooth muscle and blocks these channels in electrophysiological experiments (Knaus et al., 1994b). These results suggested that paxilline blocked maxi-K channels at a site distinct from the ChTX binding site located near the external entrance to the pore. Here we have examined block of the cloned alpha subunit (slo) of the maxi-K channel in excised membrane patches after internal application of paxilline. Paxilline caused a reversible inhibition of channel currents with slow washout kinetics. In the presence of 10 μM intracellular calcium, paxilline blocked currents elicited by brief voltage pulses with a Ki of 1.9 nM and a Hill coefficient near one. Changing the internal calcium by ten fold caused a two to three fold change in the Ki for paxilline block, with less block occurring at high calcium concentrations. Paxilline reduced the maximum of the conductance-voltage relation in a calcium-sensitive manner with less block occurring at high calcium concentrations, and caused a 20 mV depolarizing shift in the midpoint for channel opening. The time-course of relief of paxilline block by elevated calcium was more rapid than washout of paxilline suggesting an allosteric interaction between calcium and paxilline. Copyright © 1996 Elsevier Science Ltd

Section snippets

Channel expression

Mslo19, a cDNA encoding a maxi-K channel alpha subunit, was cloned from mouse brain as a naturally occurring splice variant with a full length open reading frame (GenBank locus #MMU09383) (Pallanck and Ganetzky, 1994), and 940 base pairs were removed from the 5′ region of the clone up to the second potential initiation codon. The remaining cDNA fragment (mslo19Δ940) was inserted into an expression vector (pcDNA3) following a consensus site for translation initiation. RNA was transcribed using

Time-course of channel block by paxilline

The time-course of paxilline block of mslo channels was examined using inside-out patches excised from Xenopus oocytes expressing mslo. Fig. 2 shows the current amplitudes recorded in response to pulses to +100 mV, once per min, in the presence of 1 μM internal calcium. Paxilline applied at 1 and 3 nM caused reversible reductions in current amplitudes. After washout of paxilline, the current did not recover completely to control levels, but repeated applications of the same drug concentration

DISCUSSION

Paxilline is a member of a group of structurally related indole diterpenes that are among the most potent non-peptidyl blockers of high-conductance, calcium-activated potassium channels. In ligand binding experiments, paxilline enhances binding of charybdotoxin to maxi-K channels in bovine aortic smooth muscle with a K12 of 170 nM (Knaus et al., 1994b). Paxilline also blocked maxi-K channels from bovine smooth muscle in electrophysiological experiments with a Ki of less than 10 nM. In the

Acknowledgements

We thank J. Liu for preparing RNA and oocytes, J. Herrington and R. Bookman for IGOR XOPs used to control data acquisition, and G. Kaczorowski comments on the manuscript.

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