TETRAETHYLAMMONIUM BLOCK OF SLOWPOKE CALCIUM-ACTIVATED POTASSIUM CHANNELS EXPRESSED IN XENOPUS-OOCYTES - EVIDENCE FOR TETRAMERIC CHANNEL FORMATION

Unitary currents were recorded from inside-out membrane patches pulled from Xenopus oocytes that had been injected with RNA transcribed from a cDNA encoding the Drosophila maxi-K channel (Slowpoke). Site-direct ed mutagenesis was used to make cDNAs encoding channel subunits with s ingle amino acid substitutions (Y308V and C309P). The extracellular si de of the patch was exposed to tetraethylammonium (TEA) in the pipette solution; unitary currents in the presence of TEA were compared with currents in the absence of TEA to compute the inhibition. Amplitude di stributions were fit by P functions to estimate the blocking and unblo cking rate constants. For wild-type channels, TEA blocked with an appa rent K-d of 80 mu M at 0 mV and sensed 0.18 of the membrane electric f ield; the voltage dependence lay entirely in the blocking rate constan t. TEA blocked currents through C309P channels with a similar affinity to wild-type at 0 mV, but this was not voltage-dependent. Currents th rough Y308V channels were very insensitive to any block by TEA; the ap parent K-d at 0 mV was 26 mM and the blockade sensed 0.18 of the elect ric field. Oocytes injected with a mixture of RNAs encoding wild-type and Y308V channels showed unitary currents of four discrete amplitudes in the presence of 3 mM TEA; at 40 mV these corresponded to inhibitio ns of approximately 80%, 55%, 25% and 10%. These values agreed well wi th these expected for inhibition by TEA of currents through channels c ontaining 3, 2, 1 and 0 tyrosine residues at the channel mouth, assumi ng that a tyrosine residue from each of four subunits contributes equa lly to the binding of the TEA ion. This indicates that Slowpoke channe ls form as tetramers.