The neutral, hydrophobic isoleucine at position I521 in the extracellular S4 domain of hERG contributes to channel gating equilibrium.

The human ether-a-go-go related (hERG) potassium channel has unusual functional characteristics in that the rates of channel activation and deactivation are much slower than inactivation, which is attributed to specific structural elements within the NH2 terminus and the S1-S4 voltage-sensing domains (VSD). Although the charged residues in the VSD have been extensively modified and mutated as a result, the role and importance of specific hydrophobic residues in the S4 has been much less explored in studies of hERG gating. We found that charged, but not neutral or hydrophobic, amino acid substitution of isoleucine 521 at the outer end of the S4 transmembrane domain resulted in channels activating at much more negative voltages associated with a marked hyperpolarization of the conductance-voltage (G-V) relationship. The contributions of different physicochemical properties to this effect were probed by chemical modification of channels substituted with cysteine at position I521. When positively charged reagents including tetramethyl-rhodamine-5-maleimide (TMRM), 1-(2-maleimidylethyl)-4-[5-(4-methoxyphenyl)oxazol-2-yl] pyridinium methane-sulfonate (PyMPO), [2-(trimethylammonium)ethyl] methanethiosulfonate chloride (MTSET), and 2-aminoethyl methanethiosulfonate hydrobromide (MTSEA) were bound to the cysteine, I521C channels activated at more negative membrane potentials. To examine the contributions to hERG gating of other residues at the outer end of S4 (520-528), we performed a cysteine scan combined with MTSET modification. Only L520C, along with I521C, shows a substantial hyperpolarizing shift of the G-V relationship upon MTSET modification. The data indicate that the neutral, hydrophobic residue I521 at the extracellular end of S4 is critical for stabilizing the closed conformation of the hERG channel relative to the open state and by comparison with Shaker supports the alignment of hERG I521 with Shaker L361.