Voltage-gated K+ (Kv) are tetramers of α-subunits each consisting of 6 transmembrane segments (S1-S6) and a cytoplasmic N- and C-terminus. The S5-S6 segments of each subunit assemble to generate the central pore while the S1-S4 segments form the voltage-sensing domains. The PXP motif in the middle of S6 provides a degree of flexibility to the bottom half of the S6 segment which is necessary for channel gating. This region is also critical for the interaction with channel blockers. Based on sequence homology, eight Shaker-related Kv subfamilies have been identified: Kv1-Kv6, Kv8-Kv9. The silent (KvS) subunits (Kv5-Kv9) cannot form homotetramers but assemble with Kv2 subunits into Kv2/KvS heterotetramers that display unique biophysical properties. KvS subunits lack the 2nd proline residue of the PXP motif which may impact on the pharmacological profile of channel blockers. We tested this hypothesis by using the Kv1.5(P511G) mutant in which the 2nd proline of the PXP motif was replaced by a glycine. Homotetrameric Kv1.5(P511G) channels were insensitive to 4-AP while heterotetrameric Kv1.5-Kv1.5(P511G) channels (stoichiometry controlled by using dimers), still displayed current inhibition. However, Kv1.5-Kv1.5(P511G) channels were significantly less sensitive displaying an IC50 values of 16 mM instead of 270μM for wild type (WT) Kv1.5. Similarly, heterotetrameric Kv2/KvS channels displayed an altered affinity for 4-AP compared to WT Kv2.1; 18 mM (IC50 for Kv2.1) inhibited 17%, 60%, 82% and 13% of Kv5.1, Kv6.3, Kv8.1 and Kv9.3-containing currents, respectively. Furthermore, the heterotetrameric Kv2/KvS channels displayed also a subtle change in the affinity for the open channel blockers quinidine and flecainide. These results suggest that the absence of a complete PXP motif in one or two out of four subunits alters the pharmacological profile. (Supported by FWO fellowships to JS and EB & grant FWO-G.0449.11N to DJS).