Functional control of heteromeric Kv2.1/6.4 channels by the voltage sensor domain of the silent Kv6.4 subunit

In the activation process of Kv channels, the S4 segment of the voltage-sensing domain (VSD) moves in the outward direction. A conserved phenylalanine in the transmembrane S2 helix of the VSD is viewed as operating as a charge transfer centre (CTC) that interacts with a positively charged arginine of the S4 helix. This phenylalanine is highly sensitive to diverse substitutions. Kv2.1 subunits can form functional homotetrameric channels on their own whereas 'silent' Kv6.4 subunits can only contribute to functional heterotetrameric channels. We used concatenated dimers of Kv2.1 and Kv6.4 subunits to define the stoichiometry and position of these subunits in functional heterotetrameric channels. Our results demonstrate that mutating the phenylalanine F273 of the Kv6.4 subunits in Kv 2.1_6.4 channels built of dimers to diverse other amino acids at the CTC affects steady-state activation only moderately whereas it strongly shifts steady-state inactivation by 40 mV toward more depolarized potentials compared to Kv2.1_6.4 wild-type channels. Mutating the Kv6.4 subunits in this heterotetramer slowed down the recovery from closed-state inactivation without impacting open-state inactivation. Moreover, results with the specific Kv2.1 blocker guangxitoxin suggest that Kv6.4 subunits may partly activate Kv2.1_6.4 channels. It is concluded that F273 in the silent Kv6.4 subunit of Kv2.1_6.4 channels has a unique role in controlling activation and the recovery from inactivation. HIGHLIGHTS: This study quantifies the functional effects of Kv6.4 mutations in Kv2.1_6.4 channels on activation and inactivation. Highly diverse mutations of the phenylalanine in the charge transfer centre of Kv6.4 reveal its unique role in Kv2.1_6.4 channels in closed state inactivation. The specific Kv2.1 blocker guangxitoxin unmasks that Kv6.4 subunits can partly activate Kv2.1_6.4 channels.