Probing the Gating of Kv10.1 Channels by MTS Reagents
Biophysical journal(2017)
摘要
Kv10.1 (Eag1) is a voltage-gated potassium channel mostly expressed in the brain and also found in many cancer types. Recently it was shown to perform voltage-dependent gating even in the absence of the continuity of the S4-S5 linker connecting the voltage-sensing domain (VSD, comprising the S1-S4 helices) and the pore domain (PD, comprising S5-S6). This linker was previously considered necessary to transfer VSD movements to the PD to induce opening. The L341 split channel missing the covalent link between the two domains produced constitutive current, while still showing some voltage-dependence. We applied an accessibility assay using membrane-impermeable methanethiosulfonate (MTS) reagents to investigate whether this altered gating was due to a change in VSD movements or in the coupling between the two domains in the split channel. The rate of state-dependent binding of MTS reagents to cysteines substituted at strategic positions in the VSD of the protein informs about the accessibility of S4 in various gating states. First we performed a cysteine scan of the S3-S4 linker to find a position sensitive to MTS application. L322C showed a −28mV G-V shift and slowed deactivation rate upon MTS application. This mutant showed very similar modification rates for the full-length and split channels suggesting that the two VSDs undergo similar conformational changes and the differences originate from altered coupling between the VSD and the PD. Based on its recently solved structure, a fundamentally different gating mechanism is suggested for Eag1, as the structure indicates several deviations from previously accepted models of other Kv channels, including a shorter S4-S5 linker, non-domain-swapped transmembrane segments and the lack of an activation gate hinge in the S6 segment. This prompted us to investigate the location of the activation gate using the intracellular application of MTS reagents. Support: KTIA_NAP_13-2-2015-0009.
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