Pore mutations in Shaker K+ channels distinguish between the sites of tetraethylammonium blockade and C-type inactivation.

1. We have studied the effect of external K+ and tetraethylammonium (TEA) on several mutants of Shaker B K+ channels with amino acid substitutions in the pore which alter TEA affinity and the rate of C-type inactivation. In all channels studied high external K+ makes C-type inactivation slower. 2. In the wild-type channel, TEA blockade is voltage dependent and produces slowing of the inactivation time course. However, in the double mutant channel (T449Y, D447E) TEA blockade, although of higher affinity, is voltage independent and does not affect the rate of C-type inactivation. 3. Mutants with a charged amino acid at position 449 (T449K and T449E) are resistant to TEA block. In these channels, C-type inactivation is also unaffected by TEA. 4. These results indicate that the sites where TEA blocks and competes with C-type inactivation can be segregated. To modulate inactivation, TEA must enter deeply into the channel mouth. These results suggest that C-type inactivation is not due to a large molecular rearrangement in the outer channel vestibule, but it is essentially produced by a conformational change restricted to a local site in the pore.