The gating charges in S4 segment are highly conserved in voltage-gated K+ channels. However, a broad range of voltage dependence for charge movement and K+ conductance activation are found, suggesting these features are determined by other motifs. The extracellular S3-S4 linker shows family-related conservation. We studied the influence of residues 353-361 of that region on the voltage dependence of both gating charges movement (Q-Vs) and K+-conductance activation (G-Vs) in Shaker K+-channel. Remarkably, hydrophilic mutations in L358 and L361 produce strong shifts of both Q-Vs and G-Vs to more negative voltages. The Q-V is shifted more than −80-mV inL361R. We scanned with mutagenesis L358 (L358X) and L361 (L361X) with different amino acids (AA) and measured the mid-points of Q-V curves (Vmed) and G-V curves (V0.5). We plotted those values with several AA scales and took their coefficient of determination from a linear regression (R2). For L358X, Vmed were correlated with the residue tendency to be in a transmembrane segment (R2=0.72) and V0.5 by the hydrophobic surface area of the residue (R2=0.66). For L361X, Vmed were correlated with the residue tendency to be buried in the protein (R2=0.86) and V0.5 by the hydration potential of the residue (R2=0.66). By fitting Q-Vs to a three-state sequential model we find V0 and V1 as the voltage dependence of two simplified steps during VSD activation. The voltage sensor (VS) coupling to the pore domain (PD) was accessed by plotting V1, the last step, with V0.5 for L358X (R2=0.68) and L361X (R2=0.91). V0.5 changes in both cases are not well correlated with V0 (R2<0.45). Our data show that voltage dependence and VS-to-PD coupling can be dramatically changed by single mutations in the S3-S4 linker, and these changes cannot be explained by using well known AA hydrophobicity scales. Support:NIH-GM030376.