Lu T et al. (2001), Permeant ion-dependent changes in gating of Kir...

XB-ART-8138

J Gen Physiol 2001 Nov 01;1185:509-22. doi: 10.1085/jgp.118.5.509.

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Permeant ion-dependent changes in gating of Kir2.1 inward rectifier potassium channels.

Lu T , Wu L , Xiao J , Yang J .

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We studied the effect of monovalent thallium ion (Tl(+)) on the gating of single Kir2.1 channels, which open and close spontaneously at a constant membrane potential. In cell-attached recordings of single-channel inward current, changing the external permeant ion from K(+) to Tl(+) decreases the mean open-time by approximately 20-fold. Furthermore, the channel resides predominantly at a subconductance level, which results from a slow decay (tau = 2.7 ms at -100 mV) from the fully open level immediately following channel opening. Mutation of a pore-lining cysteine (C169) to valine abolishes the slow decay and subconductance level, and single-channel recordings from channels formed by tandem tetramers containing one to three C169V mutant subunits indicate that Tl(+) must interact with at least three C169 residues to induce these effects. However, the C169V mutation does not alter the single-channel closing kinetics of Tl(+) current. These results suggest that Tl(+) ions change the conformation of the ion conduction pathway during permeation and alter gating by two distinct mechanisms. First, they interact with the thiolate groups of C169 lining the cavity to induce conformational changes of the ion passageway, and thereby produce a slow decay of single-channel current and a dominant subconductance state. Second, they interact more strongly than K(+) with the main chain carbonyl oxygens lining the selectivity filter to destabilize the open state of the channel and, thus, alter the open/close kinetics of gating. In addition to altering gating, Tl(+) greatly diminishes Ba(2+) block. The unblocking rate of Ba(2+) is increased by >22-fold when the external permeant ion is switched from K(+) to Tl(+) regardless of the direction of Ba(2+) exit. This effect cannot be explained solely by ion-ion interactions, but is consistent with the notion that Tl(+) induces conformational changes in the selectivity filter.

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Species referenced: Xenopus laevis
Genes referenced: gnl3 kcnj2 mapt

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	Figure 1. Permeation properties of Tl+ ions. (A) Whole-cell current-voltage relations of Kir2.1 channels recorded by TEVC with either K+ or Tl+ as the external permeant ion. (B) Close-up of A to show the outward current and reversal potential. (C) Relative whole-cell current recorded at â100 mV by TEVC in external solutions containing different concentrations of K+ and Tl+. The total concentration of both ions is 100 mM. Each data point is the average of four to eight measurements. Error bars are SD.
	Figure 2. Alteration of single-channel gating by Tl+ ions. (A and B) Consecutive records of single-channel K+ (A) or Tl+ (B) currents recorded at â100 mV in a cell-attached membrane patch. The dashed lines indicate the closed level in this and all subsequent figures. Arrows in B indicate the dominant subconductance level and arrowheads mark a relatively rare subconductance level. (C and D) Open- and closed-time distributions of channels recorded with K+ (C) or Tl+ (D) as the external permeant ion. The open-time distributions were best fit by a single exponential and the closed-time distributions by three exponentials. The time constants of the fits are indicated. (E) Voltage dependence of the open-time with either K+ or Tl+ as the permeant ion (n = 4â6). (F and G) Voltage dependence of the closed times with either K+ (F) or Tl+ (G) as the permeant ion (n = 4â12). Error bars are SD and are smaller than the symbols in some cases.
	Figure 3. Slow decay of single-channel Tl+ current. (A) 12 selected single-channel current events that exhibit a slow decay are aligned by their initial opening and are superimposed. The currents were recorded at â100 mV in a cell-attached membrane patch with Tl+ as the external permeant ion. (B) The dotted line shows the ensemble average of the traces in A. The decay phase is fitted by a single exponential function with a time constant of 2.7 ms (solid line).
	Figure 4. C169V mutation abolishes the slow decay of single-channel current and subconductance level. (A) Representative single-channel Tl+ currents recorded at â100 mV from a C169V mutant channel in a cell-attached membrane patch. Notice the lack of decay of single-channel current and subconductance level. (B) Open- and closed-time distributions of channels recorded with Tl+ as the external permeant ion. The open-time distribution was best fit by a single exponential and the closed-time distributions by three exponentials. The time constants of the fits are similar to those for the wild-type channel (Fig. 2 D).
	Figure 5. Tl+ must interact with three or four C169 thiolate groups to induce the slow decay of single-channel current and subconductance level. The traces show representative cell-attached single-channel Tl+ currents recorded at â100 mV from four different types of channels. The channels are formed by tandem tetramers containing one to three C169V mutant subunits: WT-C169V-C169V-C169V (CVVV), WT-WT-C169V-C169V (CCVV), WT-C169V-WT-C169V (CVCV), and WT-C169V-WT-WT (CVCC).
	Figure 6. Comparison of Ba2+ blockage of K+ and Tl+ currents. (A and B) Blockage by external Ba2+ of whole-cell K+ (A) or Tl+ (B) current recorded by TEVC. Currents were recorded from the same oocyte and were evoked by a test pulse to â100 mV from a holding potential of 0 mV. The dashed lines indicate the zero current level. Notice the difference in time scale in the two figures. (C) Voltage dependence of the apparent Kd of Ba2+ with either K+ or Tl+ as the external permeant ion (n = 5â6).
	Figure 7. Blockage of single-channel K+ or Tl+ current by external Ba2+. (A and B) Single-channel K+ or Tl+ current were recorded at â100 mV from two separate cell-attached membrane patches with 10 Î¼M (A) or 300 Î¼M (B) Ba2+ in the pipette solution. The arrows in the single-channel traces and in the closed-time distributions indicate Ba2+ blocked states. The dashed lines indicate the zero current level. The current record in A was filtered at 100 Hz and digitized at 2 KHz.
	Figure 8. Tl+ speeds up dissociation of Ba2+ from the channel. The Ba2+ unblocking rate determined from single-channel recordings is plotted against the membrane potential with either K+ or Tl+ as the external permeant ion (n = 3â9).
	Figure 9. Models of Tl+ interaction with the thiolate groups of C169 and openâ close transitions of channels in the presence external Tl+. (A) The schematics represent cross sections of the pore at the position of C169. Under our recording conditions, the external and internal permeant ion species are Tl+ and K+, respectively. When the channel is closed, due to conformational changes in the selectivity filter, the cavity is equilibrated with the internal solution and is presumably occupied by K+, which interacts weakly with the thiolate groups (I). As Tl+ permeates the cavity immediately following channel opening, it induces a reorientation of the thiolate groups and seeks to form an optimal coordination (II). This process triggers a slow conformational change of the pore, such as a constriction (but the exact nature of the conformational change is unclear). Two models are proposed for the subsequent interaction between Tl+ and the thiolate groups. In one case (III), the permeating Tl+ is optimally coordinated by all four thiolate groups, but its residence time is <50 ns. In another case (IV), a Tl+ is optimally and tightly coordinated by three thiolate groups, and the cavity is sufficiently wide to permit Tl+ ions to flow by the bound Tl+. (B) A qualitative model of transitions among the proposed closed and open states in the presence of external Tl+ (discussion). (C) An idealized single-channel Tl+ current trace. The two predominant types of channel openings, Cf to Of and Cs to Os, are preceded by two and one closed states, respectively.

References [+] :

Baukrowitz, Use-dependent blockers and exit rate of the last ion from the multi-ion pore of a K+ channel. 1996, Pubmed