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bioRxiv · 10.1101/2022.12.15.520511

State-dependent photocrosslinking unveiled the role of intersubunit interfaces on Ca2+ activating mechanisms of the BK channel

Gonzalez-Hernandez, A. J.·Rivero-Perez, B.·Bartolome-Martin, D.·Alvarez de la Rosa, D.·Plested, A. J. R.·Giraldez, T.
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Abstract

BK channels are high-conductance potassium channels that are activated by voltage and Ca2+. The pore-forming -subunits form homotetramers including a membrane-spanning domain and a cytosolic domain with a tandem of RCK-like domains (RCK1 and RCK2) per subunit. The eight RCKs compose high affinity Ca2+-binding sites that drive channel activation. Full-length Cryo-EM structures revealed intersubunit interactions between the RCK domains. Asparagine 449 (N449, human BK channel) is located at the RCK1 domain and coordinates Ca2+ in the RCK2 of the adjacent subunit. In addition, two Arginines (R786 and R790) of one RCK2 and Glutamate 955 of the adjacent subunit constitute an additional interaction interface. Functional studies on these residues showed that these two interfaces are crucial in Ca2+ sensitivity. To detect structural rearrangements induced by Ca2+ during channel activation, we took advantage of the photoactivatable unnatural amino acid p-benzoyl-L-phenylalanine (BzF). Functional channels were obtained with this amino acid inserted at 11 positions. N449 and R786 positions (N449BzF and R786BzF respectively). UV-induced photocrosslinking led to Ca2+ dependent and voltage-independent effects in both mutants. N449BzF showed a steady-state current reduction at saturating Ca2+ concentrations. Our data shows that this effect mainly relies on full occupancy of the RCK1 Ca2+ binding site, since mutation of this site abolished the effect. The R786BzF construct showed a substantial potentiation of the current in the absence of Ca2+. In this case, photocrosslinking seems to favor the activation of the channel by voltage. Overall, these results suggest mobile interfaces between RCK domains are key to BK channel activation.

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Gonzalez-Hernandez, A. J., Rivero-Perez, B., Bartolome-Martin, D., Alvarez de la Rosa, D., Plested, A. J. R., Giraldez, T.. 2022-12-18. State-dependent photocrosslinking unveiled the role of intersubunit interfaces on Ca2+ activating mechanisms of the BK channel. https://doi.org/10.1101/2022.12.15.520511

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