Abstract

In Shaker K+ channels depolarization displaces outwardly the positively charged residues of the S4 segment. The amount of this displacement is unknown, but large movements of the S4 segment should be con strained by the length and flexibility of the S3-S4 Linker. To investigate the role of the S3-S4 linker in the ShakerH4 Delta(6-46) (Shaker Delta) K+ channel activation, we constructed S3-S4 linker deletion mutants. Using macro-patches of Xenopus oocytes, we tested three constructs: a deletion mutant with no linker (0 aa linker), a mutant containing a linker 5 amino acids in length, and a 10 amino acid linker mutant. Each of the three mutants tested yielded robust K+ currents. The half-activation voltage was shifted to the right along the voltage axis, and the shift was +45 mV in the case of the 0 aa linker channel. In the 0 aa linker, mutant deactivation kinetics were sixfold slower than in Shaker Delta. The apparent number of gating charges was 12.6 +/- 0.6 e(o) in Shaker Delta, 12.7 +/- 0.5 in 10 aa linker, and 12.3 +/- 0.9 in 5 aa linker channels, but it was only 5.6 +/- 0.3 e(o) in the 0 aa linker mutant channel. The maximum probability of opening (P-o(max)) as measured using noise analysis was not altered by the linker deletions. Activation kinetics were most affected by linker deletions; at 0 mV, the 5 and 0 aa linker channels' activation time constants were 89x and 45x slower than that of the Shaker Delta K+ channel, respectively. The initial lag of ionic currents when the prepulse was varied from -130 to -60 mV was 0.5, 14, and 2 ms for the 10, 5, and 0 aa linker mu rant channels, respectively These results suggest that: (a) the 34 segment moves only a short distance during activation since an S3-S4 linker consisting of only 5 amino acid residues allows for the total charge displacement to occur; and (b) die length of tl-le S3-S4 linker plays an important role in setting Shaker Delta channel activation and deactivation kinetics.