Research ArticlePHARMACOLOGY

Molecular overlap in the regulation of SK channels by small molecules and phosphoinositides

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Science Advances  10 Jul 2015:
Vol. 1, no. 6, e1500008
DOI: 10.1126/sciadv.1500008
  • Fig. 1 Stabilization of IDF of SK2-a channel by NS309.

    (A) The IDF (dashed line) is highly flexible in the absence of NS309, and its structure could not be determined by x-ray crystallography. (B) E404-R471 distance from MD simulation in the absence (black) and presence (red) of NS309. (C) The IDF becomes well structured in the presence of NS309. NS309 forms contacts with F410. In addition, the E404-K75 salt bridge is promoted through a hydrogen bond between NS309 and K75. (D) The E404-K75 distance from MD simulations in the absence (black) and presence (red) of NS309. (E) Dose-response curves of channel reactivation by Ca2+ in the absence (solid line, filled symbols) and presence (dashed line, open symbols) of 3 μM NS309 of the wild-type (WT)/WT (SK2-a/CaM) channel. (F) NS309 increases the apparent affinity for Ca2+ of SK2-a channels.

  • Fig. 2 The effect of NS309 on PIP2 interactions with SK2-a channel.

    (A) A representative snapshot from MD simulations of PIP2 (P4 and P5 in orange and oxygens in red) binding to CaM (salmon) and SK2-a channel (gray) in the absence of NS309. (B) Summarized probability of forming salt bridges between PIP2 head and positively charged residues in the absence of NS309. (C) A representative snapshot from MD simulations of PIP2 (P4 and P5 in orange and oxygens in red) binding to CaM and SK2-a channel (gray) in the presence of NS309 (yellow). Salt bridges are shown as blue dashed lines with distances in angstrom. (D) Summarized probability of forming salt bridges between the PIP2 head and positively charged residues in the presence of NS309.

  • Fig. 3 The effect of NS309 on PIP2 sensitivity of the SK2-a channel.

    (A) Raw current traces before (black) and after (red) application of poly-K in the presence of 2 μM Ca2+. After depletion of endogenous PIP2, 2 μM Ca2+ and 3 μM NS309 (green) could not induce any measurable current increase. (B) Dose-dependent channel reactivation by exogenous diC8-PIP2 at the indicated concentrations, in the presence of 3 μM NS309, after depletion of the endogenous PIP2 by poly-K. (C) Dose-response curves of channel reactivation by exogenous diC8-PIP2 in the absence (solid line) and presence (dashed line) of 3 μM NS309 on WT/WT (SK2-a/CaM) channel. (D) NS309 increases the apparent affinity for diC8-PIP2 of SK2-a channels.

  • Fig. 4 The role of PIP2 in the effect of NS309.

    (A) Mutant channels that show decreased apparent affinity for diC8-PIP2. (B) The same group of mutations also decreases potency of NS309. (C) A clear correlation between PIP2 sensitivity and NS309 potency on the mutant channels (R = 0.981). (D) A simplified scheme for SK2-a channel activation, with Ca2+ binding to CaM as the first step and PIP2-mediated coupling to channel opening as the second step. PIP2 does not activate the channel in the absence of Ca2+. NS309 potentiates channel activity through an enhancement of function by strengthening channel-PIP2 interactions.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/1/6/e1500008/DC1

    Fig. S1. The influence of NS309 on the conformation of IDF.

    Fig. S2. The influence of PIP2 on the conformation of the IDF.

    Fig. S3. The PIP2-channel interaction is affected by NS309.

    Fig. S4. The effectiveness of NS309 on the phosphomimetic T79D CaM mutant.

    Fig. S5. The proximity of NS309 and CaM T79D mutation to the PIP2 binding site.

    Fig. S6. The PIP2-channel interaction is affected by NS309 in the context of T79D mutant.

  • Supplementary Materials

    This PDF file includes:

    • Fig. S1. The influence of NS309 on the conformation of IDF.
    • Fig. S2. The influence of PIP2 on the conformation of the IDF.
    • Fig. S3. The PIP2-channel interaction is affected by NS309.
    • Fig. S4. The effectiveness of NS309 on the phosphomimetic T79D CaM mutant.
    • Fig. S5. The proximity of NS309 and CaM T79D mutation to the PIP2 binding site.
    • Fig. S6. The PIP2-channel interaction is affected by NS309 in the context of T79D mutant.

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