Dataset Information

Hydrophobic interactions between the HA helix and S4-S5 linker modulate apparent Ca²⁺ sensitivity of SK2 channels.

ABSTRACT:

Aim

Small-conductance Ca²⁺ -activated potassium (SK) channels are activated exclusively by increases in intracellular Ca²⁺ that binds to calmodulin constitutively associated with the channel. Wild-type SK2 channels are activated by Ca²⁺ with an EC₅₀ value of ~0.3 μmol/L. Here, we investigate hydrophobic interactions between the HA helix and the S4-S5 linker as a major determinant of channel apparent Ca²⁺ sensitivity.

Methods

Site-directed mutagenesis, electrophysiological recordings and molecular dynamic (MD) simulations were utilized.

Results

Mutations that decrease hydrophobicity at the HA-S4-S5 interface lead to Ca²⁺ hyposensitivity of SK2 channels. Mutations that increase hydrophobicity result in hypersensitivity to Ca²⁺ . The Ca²⁺ hypersensitivity of the V407F mutant relies on the interaction of the cognate phenylalanine with the S4-S5 linker in the SK2 channel. Replacing the S4-S5 linker of the SK2 channel with the S4-S5 linker of the SK4 channel results in loss of the hypersensitivity caused by V407F. This difference between the S4-S5 linkers of SK2 and SK4 channels can be partially attributed to I295 equivalent to a valine in the SK4 channel. A N293A mutation in the S4-S5 linker also increases hydrophobicity at the HA-S4-S5 interface and elevates the channel apparent Ca²⁺ sensitivity. The double N293A/V407F mutations generate a highly Ca²⁺ sensitive channel, with an EC₅₀ of 0.02 μmol/L. The MD simulations of this double-mutant channel revealed a larger channel cytoplasmic gate.

Conclusion

The electrophysiological data and MD simulations collectively suggest a crucial role of the interactions between the HA helix and S4-S5 linker in the apparent Ca²⁺ sensitivity of SK2 channels.

SUBMITTER: Nam YW

PROVIDER: S-EPMC7736289 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Slow deactivation of hERG channels is critical for preventing cardiac arrhythmia yet the mechanistic basis for the slow gating transition is unclear. Here, we characterized the temporal sequence of events leading to voltage sensor stabilization upon membrane depolarization. Progressive increase in step depolarization duration slowed voltage-sensor return in a biphasic manner (τfast = 34 ms, τslow = 2.5 s). The faster phase of voltage-sensor return slowing correlated with the kinetics of pore opening. The slower component occurred over durations that exceeded channel activation and was consistent with voltage sensor relaxation. The S4-S5 linker mutation, G546L, impeded the faster phase of voltage sensor stabilization without attenuating the slower phase, suggesting that the S4-S5 linker is important for communications between the pore gate and the voltage sensor during deactivation. These data also demonstrate that the mechanisms of pore gate-opening-induced and relaxation-induced voltage-sensor stabilization are separable. Deletion of the distal N-terminus (Δ2-135) accelerated off-gating current, but did not influence the relative contribution of either mechanism of stabilization of the voltage sensor. Lastly, we characterized mode-shift behavior in hERG channels, which results from stabilization of activated channel states. The apparent mode-shift depended greatly on recording conditions. By measuring slow activation and deactivation at steady state we found the "true" mode-shift to be ∼15 mV. Interestingly, the "true" mode-shift of gating currents was ∼40 mV, much greater than that of the pore gate. This demonstrates that voltage sensor return is less energetically favorable upon repolarization than pore gate closure. We interpret this to indicate that stabilization of the activated voltage sensor limits the return of hERG channels to rest. The data suggest that this stabilization occurs as a result of reconfiguration of the pore gate upon opening by a mechanism that is influenced by the S4-S5 linker, and by a separable voltage-sensor intrinsic relaxation mechanism.

Dataset Information

Hydrophobic interactions between the HA helix and S4-S5 linker modulate apparent Ca²⁺ sensitivity of SK2 channels.

Aim

Methods

Results

Conclusion

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets

Dataset Information

Hydrophobic interactions between the HA helix and S4-S5 linker modulate apparent Ca2+ sensitivity of SK2 channels.

Aim

Methods

Results

Conclusion

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets

Hydrophobic interactions between the HA helix and S4-S5 linker modulate apparent Ca²⁺ sensitivity of SK2 channels.