The concurrent mutation of three hydrophobic residues (V369I, I372L, and S376T) in the N-terminus of the S4 segment of Shaker K+ channel slows down the kinetic of channel activation by a ∼6-fold factor and shifts the conductance-voltage (G-V) curve to 115 mV (Smith-Maxwell et al., 1998). We sought to determine the effect of the ILT mutations in heterotetrameric concatemerized channels with different stoichiometries to gain insights into the mechanisms of action of the ILT mutations. The channels were expressed in Xenopus oocytes and studied using cut-open oocyte voltage clamp and patch clamp techniques. The G-V curve of a concatemer with the ILT mutations in only one subunit (wtILT/3wt) is centered at ∼8 mV compared with ∼115 mV for the homotetramers Shaker ILT, while the homotetramer concatemerized Shaker zH4 Δ(6-46) (4wt) as a V1/2 of −20 mV. The slope of the G-V curve is reduced. The kinetics of activation and deactivation are similar between 4wt and wtILT/3wt. The ILT mutations were introduced on the single wild type voltage sensor of a heterotetramer having three subunits with the four gating charges neutralized (wtILT/3mut; mut = R362Q/R365Q/R368N/R371Q). The G-V of the wtILT/3mut heterotetramer is right shifted (V1/2 ∼43 mV) compared with the wtILT/3wt and its slope is even more reduced. The number of closed states estimated from the Cole-Moore shift with an exponential function raised to a power n was slightly higher for wtILT/3wt than that of 4wt [9.5±2(9) vs. 7.5±1.5(8), P=0.05]. However, wt/3mut and wtILT/3mut have the same estimates [n=1.58±0.4(5) vs. 1.8±0.7(7), P=0.5]. wtILT/3wt has the same single channel conductance than Shaker ILT. We conclude that a single ILT wild type subunit is not sufficient for fully modifying the concerted step as in homotetramers. (Support: NIHGM30376 and NSERC fellowship to DGG)