Sequential Electrostatic Interactions between E160 in S2 and Arginines in S4 During Voltage Dependent Activation of Kv7.1 Channels

Abstract

The fourth transmembrane segment of Kv channels, S4, contains a series of positively charged residues that imparts voltage sensitivity to the channel. Because the insertion of a highly charged peptide into a hydrophobic lipid environment is energetically unfavorable, electrostatic interactions with countercharges in the protein and phospholipids are required to lower this energy barrier. However, once the protein has been inserted into the membrane, what further role do these interactions play? In functional channels, electrostatic interactions are assumed to stabilize voltage sensor movement from a resting to an activated conformation. Although this assumption is at the crux of many models of voltage dependent gating, experimental evidence specifically examining these interactions in functional channels is incomplete. Here, we demonstrate in Kv7.1 channels that the first glutamate in S2, E160 (E1), form state dependent electrostatic interactions with arginines in S4. We used charged MTS reagents to directly probe the environment around E1 after mutating E1 to cysteine. We found that MTSES- but not MTSET+ modifies E1C, suggesting a positively charged environment around E1. Mutations neutralizing or reversing the charge of the first or fourth arginine in S4 (R1 or R4) change the polarity of the environment around E1C such that MTSET+ modifies E1C in the presence of these secondary mutations. Therefore, R1 and R4 both contribute to the positive electrostatic environment around E1. Moreover, MTSET+ modification of E1C with R1E could only occur at hyperpolarizing voltages but not at depolarizing voltages, suggesting that R1 is proximal to E1 only at the resting state but moves distally at the activated state. Overall, our data is consistent with a mechanism where arginines interact sequentially with E1 as S4 moves from a resting to an activated conformation.