The S4 segment of the jellyfish (Polyorchis penicillatus) Shaker channel jShak1 contains only six positively charged motifs. All other Shaker channels, including the jellyfish Shaker channel jShak2, have seven charges in this segment. Despite their charge differences, both these jellyfish channels produce currents with activation and inactivation curves shifted by approximately +40 mV relative to other Shaker currents. Adding charge without changing segment length by mutating the N-terminal

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... of jShak1 S4 does not have a pronounced effect on channel activation properties. Adding the positively charged motif RIF on the N-terminal side of K294 (the homologue of K374 in Drosophila Shaker, which is a structurally critical residue) produced a large positive shift in both activation and inactivation without altering the slope of the activation curve of the channel. When IFR was added to the other side of K294, there was a small negative shift in activation and fast inactivation of the channel was prevented. Our results demonstrate that K294 divides the S4 segment into functionally different regions and that the voltage threshold for activation and inactivation of the channel is not determined by the total charge on S4.