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Structural implications of weak Ca2+ block in Drosophila cyclic nucleotide-gated channels.

Lam YL, Zeng W, Derebe MG, Jiang Y - J. Gen. Physiol. (2015)

Bottom Line: The selectivity filter of the Drosophila CNG channel is similar to that of most other CNG channels except that it has a threonine at residue 318 instead of a proline.Moreover, mutating the corresponding threonine (T318) to proline in Drosophila CNG channels increased Ca(2+) block by 16 times.These results imply that a simple replacement of a threonine for a proline in Drosophila CNG channels has likely given rise to a distinct selectivity filter conformation that results in weak Ca(2+) block.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Physiology and Howard Hughes Medical Institute, University of Texas Southwestern Medical Center, Dallas, TX 75390.

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Ion conduction and Ca2+ block of NaK2CNG-Dm. (A; left) Sample traces of single-channel recordings of NaK2CNG-Dm and NaK2CNG-E at ±100 mV. (Right) Histogram of single-channel current at −100 mV. (B) Ion selectivity and conductance of NaK2CNG-Dm and NaK2CNG-E are compared in I-V plot. Error bars are the mean ± SEM of five independent measurements. (C) Sample traces of single-channel recordings at −100 mV at different external (bath) Ca2+ concentrations. (D) Fraction of unblocked current (I/Imax) at −100 mV as a function of Ca2+ concentration. Data are fitted into a Hill equation, with Ki of 4 ± 0.6 mM and a Hill coefficient n of 0.52 for NaK2CNG-Dm and Ki of 59 ± 10 µM and n of 0.98 for NaK2CNG-E. Error bars are mean ± SEM from five measurements.
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fig4: Ion conduction and Ca2+ block of NaK2CNG-Dm. (A; left) Sample traces of single-channel recordings of NaK2CNG-Dm and NaK2CNG-E at ±100 mV. (Right) Histogram of single-channel current at −100 mV. (B) Ion selectivity and conductance of NaK2CNG-Dm and NaK2CNG-E are compared in I-V plot. Error bars are the mean ± SEM of five independent measurements. (C) Sample traces of single-channel recordings at −100 mV at different external (bath) Ca2+ concentrations. (D) Fraction of unblocked current (I/Imax) at −100 mV as a function of Ca2+ concentration. Data are fitted into a Hill equation, with Ki of 4 ± 0.6 mM and a Hill coefficient n of 0.52 for NaK2CNG-Dm and Ki of 59 ± 10 µM and n of 0.98 for NaK2CNG-E. Error bars are mean ± SEM from five measurements.

Mentions: Giant liposome patch clamp was used to assay the ion permeation and Ca2+-blocking properties of the NaK2CNG-Dm channel. Similar to NaK2CNG-E, NaK2CNG-Dm has high open probability and exhibits some subconductance states (Fig. 4 A). Under bi-ionic condition with 150 mM KCl in bath and 150 mM NaCl in pipette, NaK2CNG-Dm has a single-channel conductance of ∼56 pS for inward K+ current at −100 mV, much smaller than that of NaK2CNG-E (∼100 pS) measured previously under the same condition (Derebe et al., 2011). Interestingly, NaK2CNG-Dm is weakly selective for K+ with a reversal potential of 25 mV, corresponding to PK/PNa of ∼2.7 (Fig. 4 B). This is in contrast to NaK2CNG-E, which was reported previously to be nonselective (Derebe et al., 2011). The Ca2+ block of NaK2CNG-Dm was measured with various concentrations of Ca2+ added to the external side of the channel (bath solution). As shown in Fig. 4 (C and D), Ca2+ only weakly blocked the monovalent ion current in NaK2CNG-Dm with a Ki of ∼4 ± 0.6 mM, which is in stark contrast with the Ki of 59 ± 10 µM in NaK2CNG-E.


Structural implications of weak Ca2+ block in Drosophila cyclic nucleotide-gated channels.

Lam YL, Zeng W, Derebe MG, Jiang Y - J. Gen. Physiol. (2015)

Ion conduction and Ca2+ block of NaK2CNG-Dm. (A; left) Sample traces of single-channel recordings of NaK2CNG-Dm and NaK2CNG-E at ±100 mV. (Right) Histogram of single-channel current at −100 mV. (B) Ion selectivity and conductance of NaK2CNG-Dm and NaK2CNG-E are compared in I-V plot. Error bars are the mean ± SEM of five independent measurements. (C) Sample traces of single-channel recordings at −100 mV at different external (bath) Ca2+ concentrations. (D) Fraction of unblocked current (I/Imax) at −100 mV as a function of Ca2+ concentration. Data are fitted into a Hill equation, with Ki of 4 ± 0.6 mM and a Hill coefficient n of 0.52 for NaK2CNG-Dm and Ki of 59 ± 10 µM and n of 0.98 for NaK2CNG-E. Error bars are mean ± SEM from five measurements.
© Copyright Policy - openaccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4555469&req=5

fig4: Ion conduction and Ca2+ block of NaK2CNG-Dm. (A; left) Sample traces of single-channel recordings of NaK2CNG-Dm and NaK2CNG-E at ±100 mV. (Right) Histogram of single-channel current at −100 mV. (B) Ion selectivity and conductance of NaK2CNG-Dm and NaK2CNG-E are compared in I-V plot. Error bars are the mean ± SEM of five independent measurements. (C) Sample traces of single-channel recordings at −100 mV at different external (bath) Ca2+ concentrations. (D) Fraction of unblocked current (I/Imax) at −100 mV as a function of Ca2+ concentration. Data are fitted into a Hill equation, with Ki of 4 ± 0.6 mM and a Hill coefficient n of 0.52 for NaK2CNG-Dm and Ki of 59 ± 10 µM and n of 0.98 for NaK2CNG-E. Error bars are mean ± SEM from five measurements.
Mentions: Giant liposome patch clamp was used to assay the ion permeation and Ca2+-blocking properties of the NaK2CNG-Dm channel. Similar to NaK2CNG-E, NaK2CNG-Dm has high open probability and exhibits some subconductance states (Fig. 4 A). Under bi-ionic condition with 150 mM KCl in bath and 150 mM NaCl in pipette, NaK2CNG-Dm has a single-channel conductance of ∼56 pS for inward K+ current at −100 mV, much smaller than that of NaK2CNG-E (∼100 pS) measured previously under the same condition (Derebe et al., 2011). Interestingly, NaK2CNG-Dm is weakly selective for K+ with a reversal potential of 25 mV, corresponding to PK/PNa of ∼2.7 (Fig. 4 B). This is in contrast to NaK2CNG-E, which was reported previously to be nonselective (Derebe et al., 2011). The Ca2+ block of NaK2CNG-Dm was measured with various concentrations of Ca2+ added to the external side of the channel (bath solution). As shown in Fig. 4 (C and D), Ca2+ only weakly blocked the monovalent ion current in NaK2CNG-Dm with a Ki of ∼4 ± 0.6 mM, which is in stark contrast with the Ki of 59 ± 10 µM in NaK2CNG-E.

Bottom Line: The selectivity filter of the Drosophila CNG channel is similar to that of most other CNG channels except that it has a threonine at residue 318 instead of a proline.Moreover, mutating the corresponding threonine (T318) to proline in Drosophila CNG channels increased Ca(2+) block by 16 times.These results imply that a simple replacement of a threonine for a proline in Drosophila CNG channels has likely given rise to a distinct selectivity filter conformation that results in weak Ca(2+) block.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Physiology and Howard Hughes Medical Institute, University of Texas Southwestern Medical Center, Dallas, TX 75390.

Show MeSH
Related in: MedlinePlus