Molecular Mechanisms of Ion Channel Function 
"chain." Deletion mutations in this region accel- 
erated the inactivation process by shortening the 
chain part of the sequence. We tested this model 
further by applying a solution containing the free 
synthetic inactivation particle to the inside face 
of mutant channels that did not inactivate. In the 
presence of the synthetic inactivation particle, 
the mutant channels regained inactivation, con- 
sistent with the ball and chain mechanism. We 
are now using synthetic inactivation particles and 
mutant channels to examine the detailed struc- 
tural requirements for inactivation. 
Shaker potassium channels also exhibit a 
slower inactivation process. It can be seen both in 
wild-type channels and after the faster inactiva- 
tion process has been removed by mutagenesis. 
The slow inactivation does not require intact fast 
inactivation. Although it also does not involve re- 
arrangement of charge in the membrane, this 
slower inactivation seems to involve a mecha- 
nism different from the fast process. In collab- 
oration with Kathleen Choi and Gary Yellen 
(HHMI, the Johns Hopkins University) we have 
found that the slow inactivation is affected by 
external agents, suggesting that the conforma- 
tional changes for this process involve external 
structures. 
The slow inactivation process occurs by greatly 
different rates in variants of the Shaker channel 
with differences in structure at the carboxyl end 
of the protein. We have made mutations in both 
of these variants and have localized to a single 
hydrophobic amino acid in a membrane-span- 
ning region of the channel molecule the differ- 
ence responsible for the slow inactivation differ- 
ences. Other amino acid substitutions at this 
position have dramatic effects on gating, with 
larger hydrophobic amino acids leading to slower 
inactivation. We are currently investigating this 
process further. 
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