Zhang XD et al. (2006), Roles of K149, G352, and H401 in the channel fu...

XB-ART-535

J Gen Physiol 2006 Apr 01;1274:435-47. doi: 10.1085/jgp.200509460.

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Roles of K149, G352, and H401 in the channel functions of ClC-0: testing the predictions from theoretical calculations.

Zhang XD, Li Y, Yu WP, Chen TY.

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The ClC family of Cl(-) channels and transporters comprises membrane proteins ubiquitously present in species ranging from prokaryotes to mammals. The recently solved structures of the bacterial ClC proteins have provided a good model to guide the functional experiments for the eukaryotic Cl(-) channels. Theoretical calculations based on the bacterial ClC structures have identified several residues critical for the Cl(-) binding energy in the Cl(-) transport pathway. It was speculated that the corresponding residues in eukaryotic Cl(-) channels might play similar roles for the channel functions. In this study, we made a series of mutations in three such residues in eukaryotic ClC Cl(-) channels (K149, G352, and H401 in ClC-0) and studied the functional consequences on the channel properties. A cysteine modification approach was also employed to evaluate the electrostatic effects of the charge placed at these three positions. The experimental results revealed that among the three residues tested, K149 plays the most important role in controlling both the gating and the permeation functions of ClC-0. On the other hand, mutations of H401 alter the channel conductance but not the gating properties, while mutations of G352 result in very little functional consequence. The mutation of K149 into a neutral residue leucine (K149L) shifts the activation curve and leads to flickery channel openings. The anion permeability ratios derived from bi-ionic experiments are also significantly altered in that the selectivity of Cl(-) over other anions is decreased. Furthermore, removing the positive charge at this position reduces and increases, respectively, the accessibility of the negatively and positively charged methane thiosulfonate reagents to the pore. The control of the accessibility to charged MTS reagents and the regulation of the anion permeation support the idea that K149 exerts an electrostatic effect on the channel function, confirming the prediction from computational studies.

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	Figure 1. Amino acid residues that were experimentally or theoretically identified to be important for the functions of ClC proteins. Residues in red, purple, magenta, and blue have been experimentally shown to be critical for the pore function of ClC-0. Residues in CPK colors are those predicted to be important based on the ClC-ec1 structure. Their corresponding residues in ClC-0 and ClC-1 are shown as labeled by the numbers 0 and 1 at the left upper corner of the nomenclature.
	Figure 2. Functional roles of G352 of ClC-0 and E417 of ClC-1. (A) Whole oocyte current recordings for the WT ClC-0 and the G352E and G352Q mutants of ClC-0. Experiments were performed in the ND96 solution using the standard voltage protocol. (B) Whole oocyte recordings for the WT ClC-1 and the E417G and E417Q mutants of ClC-1. Same recording voltage protocols and solutions as those in A. (C) Instantaneous I-V relations for the three channels shown in A. Symbols are: filled squares, WT ClC-0; open circles, G352E; open triangles, G352Q. (D) Instantaneous I-V curve for the three channels shown in B. Symbols are: filled squares, WT ClC-1; open circles, E417G; open triangles, E417Q.
	Figure 3. Permeation and gating properties of G352E of ClC-0. (A) Single channel recordings of the WT ClC-0 and the G352E mutant of ClC-0 at two voltages. (B) Single-channel i-V relationships for the WT ClC-0 (open squares) and the G352E mutant (open triangles). (C) Comparing gating parameters (Po, open probability; OR, opening rate; CR, closing rate) of WT ClC-0 (filled squares) and the G352E mutant (open circles).
	Figure 4. Gating effects of extracellular Cl− between WT ClC-0 and the H401E mutant. (A) Macroscopic current recordings of WT ClC-0 in 100 and 4 mM [Cl−]o solutions. Dotted line represents the zero-current level.(B) Macroscopic current recordings of H401E mutant of ClC-0 in 100 and 4 mM [Cl−]o solution. (C) Steady-state Po-V curves of WT ClC-0 in 100 (squares), 20 (circles), 4 (up triangles), and 1 mM (down triangles) [Cl−]o. (D) Steady-state Po-V curves of H401E mutant. Experimental conditions and symbols are the same as those in C.
	Figure 5. Single-channel i-V curves of the H401E mutant and the WT ClC-0. Real-time recording traces are compared in the top panel. The recordings were performed with nearly symmetrical Cl− solutions on both sides of the membrane (142 vs. 140 mM Cl− in the pipette and bath solutions, respectively). Vm = −100 mV. The comparison of single-channel i-V curves between WT ClC-0 and H401E mutant is shown in the bottom panel.
	Figure 6. H401E, but not G352E mutant, shows inward current rectification when comparing with the WT ClC-0. (A) Macroscopic current recording of WT ClC-0 from an excised inside-out patch. Inset, instantaneous I-V curve of WT ClC-0 from the average of five patches. Symmetrical 140 mM Cl− solutions on both sides of the membrane. (B) Macroscopic current recordings of G352E mutant in an excised inside-out patch. (C) Macroscopic current recordings of H401E mutant. Insets in B (n = 8) and C (n = 10) are the same as that in A. (D) Rectification indices of WT, G352E, and H401E mutants.
	Figure 7. Effects of K149 mutations on the ClC-0 fast gating. (A) Whole oocyte recordings of K149L, K149R, and K149A mutants. Test voltages were from +180 to −160 mV. (B) Steady-state Po-V curves (left), opening rate curves (middle), and closing rate curves (right) derived from experiments as shown in A. Symbols are: squares, WT ClC-0; up triangles, K149R; down triangles, K149A; circles, K149L. For opening and closing rate, data at voltages equal to or above −40 mV are not available because the time constant of the deactivation process cannot be reliably measured. (C) Single-channel recording traces of the K149L mutant and the WT channel at −50 mV. The 500-ms recording trace indicated by the horizontal bar is expanded to the right of each trace.
	Figure 8. Macroscopic current recordings of WT CLC-0 and the K149L and K149R mutants in excised inside-out patches under bi-ionic conditions. The pipette solution contained 140 mM Cl− while the bath solution contained the same concentration of the indicated ion. Voltages of the test pulse were from +80 mV to −160 mV in −20 mV steps, and the voltage of the tailed pulse was −100 mV. In each panel the vertical axis represents current in pA while the horizontal axis represents time in ms. The prepulse current is only shown for the final 10 ms. Dashed lines indicate the zero-current level.
	Figure 9. Effects of charge mutations of ClC-0 on the MTS modification rates of Y512C mutants. (A) MTSES modification process of Y512C in the presence of WT, G352E, H401E, or K149L background. The color codes are: black, Y512C/WT; red, Y512C/G352E; green, Y512C/H401E; blue, Y512C/K149L. (B) MTSEH modification time course of the same mutants as in A. (C) Calculated rate constants of MTSES and MTSEH modifications of the mutants shown in A and B. Note that the K149L mutation significantly alters the MTSES modification rate but not the MTSEH modification rate. Color codes of different mutants in B and C are the same as that in A. (D) Rate constants of MTSES, MTSEH, and MTSEA modifications of Y512C in the presence of various amino acids at position 149 as indicated.

References [+] :

Accardi, Secondary active transport mediated by a prokaryotic homologue of ClC Cl- channels. 2004, Pubmed