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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.

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

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Functional comparison of bovine CNGA1 and P366T mutant. (A; left) Sample traces of single-channel recordings of bovine CNGA1 and P366T at −100 mV in cell-attached configuration. (Right) Histogram of single-channel current at −100 mV. (B) Normalized macroscopic currents of wild-type bovine CNGA1 and P366T mutant as a function of [cGMP]i measured in inside-out patches at −100 mV. Data are fitted to the Hill equation. The K1/2 and n of bovine CNGA1 are 39.5 ± 3.6 µM and 1.2, respectively. The K1/2 and n of P366T are 41.2 ± 4.7 µM and 1.3, respectively. (C) I-V curves of bovine CNGA1 and P366T mutant measured from outside-out patches 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 3.5 ± 0.5 µM and n of 1.03 for bovine CNGA1 and Ki of 6.0 ± 0.7 µM and n of 1.36 for P366T. All error bars are mean ± SEM from five measurements.
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fig6: Functional comparison of bovine CNGA1 and P366T mutant. (A; left) Sample traces of single-channel recordings of bovine CNGA1 and P366T at −100 mV in cell-attached configuration. (Right) Histogram of single-channel current at −100 mV. (B) Normalized macroscopic currents of wild-type bovine CNGA1 and P366T mutant as a function of [cGMP]i measured in inside-out patches at −100 mV. Data are fitted to the Hill equation. The K1/2 and n of bovine CNGA1 are 39.5 ± 3.6 µM and 1.2, respectively. The K1/2 and n of P366T are 41.2 ± 4.7 µM and 1.3, respectively. (C) I-V curves of bovine CNGA1 and P366T mutant measured from outside-out patches 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 3.5 ± 0.5 µM and n of 1.03 for bovine CNGA1 and Ki of 6.0 ± 0.7 µM and n of 1.36 for P366T. All error bars are mean ± SEM from five measurements.

Mentions: Because threonine 318 in Drosophila was found to be the key in gauging Ca2+ block, we asked whether the equivalent residue in mammalian CNG channels is important for the block. This was tested on bovine CNGA1 channel by replacing the equivalent proline (P366) with threonine, resulting in a selectivity filter sequence of TIGETPTP. The P366T mutation in bovine CNGA1 has no obvious effect on single-channel conductance and kinetics or cGMP-regulated gating (Fig. 6, A and B). However, contrary to our prediction based on the NaK2CNG mutants as well as the mutation on Drosophila CNGA, the Pro-to-Thr replacement in bovine CNGA1 has almost no effect on Ca2+ block. Outside-out patch recordings show that mutation only results in a subtle decrease of Ca2+ affinity (Ki = 3.5 ± 0.5 µM for wild type and Ki = 6.0 ± 0.7 µM for P366T; Fig. 6, C and D), suggesting that the equivalent Thr/Pro swap in CNGA1 does not compromise the structure integrity of the filter, and the channel thereby retains its strong Ca2+ block.


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

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

Functional comparison of bovine CNGA1 and P366T mutant. (A; left) Sample traces of single-channel recordings of bovine CNGA1 and P366T at −100 mV in cell-attached configuration. (Right) Histogram of single-channel current at −100 mV. (B) Normalized macroscopic currents of wild-type bovine CNGA1 and P366T mutant as a function of [cGMP]i measured in inside-out patches at −100 mV. Data are fitted to the Hill equation. The K1/2 and n of bovine CNGA1 are 39.5 ± 3.6 µM and 1.2, respectively. The K1/2 and n of P366T are 41.2 ± 4.7 µM and 1.3, respectively. (C) I-V curves of bovine CNGA1 and P366T mutant measured from outside-out patches 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 3.5 ± 0.5 µM and n of 1.03 for bovine CNGA1 and Ki of 6.0 ± 0.7 µM and n of 1.36 for P366T. All error bars are mean ± SEM from five measurements.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4555469&req=5

fig6: Functional comparison of bovine CNGA1 and P366T mutant. (A; left) Sample traces of single-channel recordings of bovine CNGA1 and P366T at −100 mV in cell-attached configuration. (Right) Histogram of single-channel current at −100 mV. (B) Normalized macroscopic currents of wild-type bovine CNGA1 and P366T mutant as a function of [cGMP]i measured in inside-out patches at −100 mV. Data are fitted to the Hill equation. The K1/2 and n of bovine CNGA1 are 39.5 ± 3.6 µM and 1.2, respectively. The K1/2 and n of P366T are 41.2 ± 4.7 µM and 1.3, respectively. (C) I-V curves of bovine CNGA1 and P366T mutant measured from outside-out patches 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 3.5 ± 0.5 µM and n of 1.03 for bovine CNGA1 and Ki of 6.0 ± 0.7 µM and n of 1.36 for P366T. All error bars are mean ± SEM from five measurements.
Mentions: Because threonine 318 in Drosophila was found to be the key in gauging Ca2+ block, we asked whether the equivalent residue in mammalian CNG channels is important for the block. This was tested on bovine CNGA1 channel by replacing the equivalent proline (P366) with threonine, resulting in a selectivity filter sequence of TIGETPTP. The P366T mutation in bovine CNGA1 has no obvious effect on single-channel conductance and kinetics or cGMP-regulated gating (Fig. 6, A and B). However, contrary to our prediction based on the NaK2CNG mutants as well as the mutation on Drosophila CNGA, the Pro-to-Thr replacement in bovine CNGA1 has almost no effect on Ca2+ block. Outside-out patch recordings show that mutation only results in a subtle decrease of Ca2+ affinity (Ki = 3.5 ± 0.5 µM for wild type and Ki = 6.0 ± 0.7 µM for P366T; Fig. 6, C and D), suggesting that the equivalent Thr/Pro swap in CNGA1 does not compromise the structure integrity of the filter, and the channel thereby retains its strong Ca2+ block.

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