Structural implications of weak Ca2+ block in Drosophila cyclic nucleotide-gated channels.
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.
Affiliation: Department of Physiology and Howard Hughes Medical Institute, University of Texas Southwestern Medical Center, Dallas, TX 75390.Show MeSH
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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.
Affiliation: Department of Physiology and Howard Hughes Medical Institute, University of Texas Southwestern Medical Center, Dallas, TX 75390.