S4 Arginines Make Unique Contributions to Voltage Dependent Gating Due to Electrostatic Interactions and the Membrane Potential

Conserved positively charged arginines in the fourth transmembrane segment (S4) of Kv channels are responsible for imparting voltage sensitivity to the channel. There are several forces that may influence these arginines including the membrane potential and electrostatic interactions with countercharges. In Shaker channels, the first four arginines are the primary gating charges that sense the membrane potential. Kv7.1 has fewer positively charged S4 residues than Shaker, notably with the third arginine in Shaker replaced by a glutamine (Q3). Further loss of charge induced by charge reversal at R1 (R1E) in Kv7.1 results in constitutively activated channels, perhaps due to insufficient charge in S4. Consistent with this idea, introduction of a positive charge at Q3 (Q3R) can restore voltage dependent activation to R1E, suggesting that Q3R may substitute for the loss of gating charge at R1E. In a related study, we have demonstrated in Kv7.1 channels that residues corresponding to the first four arginines in Shaker channels (R1-R4) interact sequentially with the first conserved glutamate in S2 (E1) during gating. Here we show via intragenic suppression that S4 arginines also interact electrostatically with the second conserved glutamate in S2 (E2), and these electrostatic interactions play an important role in voltage sensing of S4. Therefore, a network of electrostatic interactions and the membrane potential act on S4 arginines, and the balance of these forces stabilize the conformation of the voltage sensor at different states. The combination of these interactions acts uniquely on each arginine such that each arginine plays a different role in voltage dependent gating. In Kv7.1, the first two arginines (R1, R2) stabilize the resting state while the last three charged residues (R4, H5, R6) stabilize the activated state.