In response to changes in membrane potential, voltage-dependent ion channel proteins undergo conformational rearrangements that lead to channel opening. These rearrangements move a net charge, measured as "gating current", across the membrane. Here we characterize the effects of the pharmacological blocker 4-aminopyridine on both the K+ and gating currents of wild-type and mutant Shaker K+ channels. Our results indicate that the activation of these channels involves two distinct types of structural rearrangement. In addition to independent Hodgkin and Huxley type rearrangements for each of the four subunits, which are responsible for most of the gating charge movement, Shaker channels interconvert between two quaternary conformations during activation. The transition between the two quaternary states moves about 10% of the total gating charge, and it is selectively blocked by 4-aminopyridine.