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