The voltage-gated potassium channel subunit Kv2.1 forms heterotetrameric channels with the silent subunit Kv6.4. Chimeric Kv2.1 channels containing a single transmembrane segment from Kv6.4 have been shown to be functional. However, a Kv2.1 chimera containing both S1 and S5 from Kv6.4 was not functional. Back mutation of individual residues in this chimera (to the Kv2.1 counterpart) identified four positions that were critical for functionality: A200V and A203T in S1, and T343M and P347S in S5. To test for possible interactions in Kv2.1, we used substitutions with charged residues and tryptophan for the outermost pair 203/347. Combinations of substitutions with opposite charges at both T203 and S347 were tolerated but resulted in channels with altered gating kinetics, as did the combination of negatively charged aspartate substitutions. Double mutant cycle analysis with these mutants indicated that both residues are energetically coupled. In contrast, replacing both residues with a positively charged lysine together (T203K + S347K) was not tolerated and resulted in a folding or trafficking deficiency. The nonfunctionality of the T203K + S347K mutation could be restored by introducing the R300E mutation in the S4 segment of the voltage sensor. These results indicate that these specific S1, S4, and S5 residues are in close proximity and interact with each other in the functional channel, but are also important determinants for Kv2.1 channel maturation. These data support the view of an anchoring interaction between S1 and S5, but indicate that this interaction surface is more extensive than previously proposed.