The role of the S4‐S5 linker in the Ca 2+ ‐sensitivity of the K + channel hIK1 (KCNN4).

Alanine scanning of the channel region linking the S4 and S5 transmembrane helices of the hIK channel revealed several residues critical for protein trafficking and function. Substitution of phenylalanine with alanine (F194A), produced a channel that was inactive. Placing a tryptophan at this site (F194W), produced a channel that was functional, but initial whole‐cell patch‐clamp experiments indicated that the hIK‐F194W channel could no longer be stimulated by the channel opener DC‐EBIO. We determined the properties of this S4–S5 linker mutant channel further using giant‐patch and single‐channel patch clamp electrophysiological techniques. Upon patch excision, hIK‐WT channel activity decreased ~ 70% over 5 min while the hIK‐F194W channel only decreased ~10%. In excised patches, dose response relationships to free calcium gave an EC 50 of 304 ± 35.8 nM, Hill 3.3 ± 0.4 (n = 6) for the hIK‐F194W which was shifted from an EC 50 of 1.19 ± 0.11 μM, Hill 2.8 ± 0.2 (n = 8) for hIK‐WT. After rundown, hIK‐WT channel activity could be stimulated ~ 500% by ATP or DC‐EBIO, while hIK‐F194W channel activity was only stimulated ~ 15% by either maneuver. Non‐stationary variance analysis on channel activity produced by rundown and reactivation or 0 to 10μM calcium steps suggested that the Po of hIK‐WT was ~ 0.5 while the hIK‐F194W was ~ 0.9. This observation explains the small ~10% increase in patch currents upon addition of DC‐EBIO and ATP as channels are almost maximally open in 10μM Ca 2+ . We conclude that the S4‐S5 linker region has a role in hIK channel gating and suggest that the hIK‐F194W mutant channel may be useful in dissecting the mechanisms by which ATP and Ca 2+ coordinate to regulate the activity of the hIK Ca 2+ sensitive potassium channel.