Becchetti A et al. (1999), Cyclic nucleotide-gated channels. Pore topology...

XB-ART-12418

J Gen Physiol 1999 Sep 01;1143:377-92.

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Cyclic nucleotide-gated channels. Pore topology studied through the accessibility of reporter cysteines.

Becchetti A , Gamel K , Torre V .

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In voltage- and cyclic nucleotide-gated ion channels, the amino-acid loop that connects the S5 and S6 transmembrane domains, is a major component of the channel pore. It determines ion selectivity and participates in gating. In the alpha subunit of cyclic nucleotide-gated channels from bovine rod, the pore loop is formed by the residues R345-S371, here called R1-S27. These 24 residues were mutated one by one into a cysteine. Mutant channels were expressed in Xenopus laevis oocytes and currents were recorded from excised membrane patches. The accessibility of the substituted cysteines from both sides of the plasma membrane was tested with the thiol-specific reagents 2-aminoethyl methanethiosulfonate (MTSEA) and [2-(trimethylammonium)ethyl]methanethiosulfonate (MTSET). Residues V4C, T20C, and P22C were accessible to MTSET only from the external side of the plasma membrane, and to MTSEA from both sides of the plasma membrane. The effect of MTSEA applied to the inner side of T20C and P22C was prevented by adding 10 mM cysteine to the external side of the plasma membrane. W9C was accessible to MTSET from the internal side only. L7C residue was accessible to internal MTSET, but the inhibition was partial, approximately 50% when the MTS compound was applied in the absence of cGMP and 25% when it was applied in the presence of cGMP, suggesting that this residue is not located inside the pore lumen and that it changes its position during gating. Currents from T15C and T16C mutants were rapidly potentiated by intracellular MTSET. In T16C, a slower partial inhibition took place after the initial potentiation. Current from I17C progressively decayed in inside-out patches. The rundown was accelerated by inwardly applied MTSET. The accessibility results of MTSET indicate a well-defined topology of the channel pore in which residues between L7 and I17 are inwardly accessible, residue G18 and E19 form the narrowest section of the pore, and T20, P21, P22 and V4 are outwardly accessible.

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Species referenced: Xenopus laevis
Genes referenced: cdkn1a kcnb1 myh1 nsg1 tbx2

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	Figure 4. MTSET's effect on macroscopic currents from W9C and L12C channels. (A) MTSET application on the inner side of membrane patches. (Top) MTSET's effect on W9C mutant in the closed state (c.s.). (Bottom) MTSET's effect on W9C mutant in the open state (o.s.). (B) MTSET application on the outer side of membrane patches. (Top) MTSET's effect on W9C mutant in the open state (o.s.). (Bottom) MTSET's effect on L12C mutant in the open state (o.s.).
	Figure 1. Amino acid sequence alignment of the P loop residues in channels belonging to the voltage-gated ion channel superfamily (Jan and Jan 1990). From top to bottom: sequence alignment of the a subunit of the bovine rod CNG channel (R345âS371, here numbered 1â27; Kaupp et al. 1989), the catfish olfactory CNG channel (E316âE341; Goulding et al. 1992), the Shaker voltage-gated K1 channel from Drosophila (S428âW454; Tempel et al. 1987), the Kv2.1 voltage-gated K1 channel from rat brain (K358âK382; Frech et al. 1989), and the KcsA K1 channel from Streptomyces lividans (KcsA, T61âW87; Schrempf et al. 1995).
	Figure 2. MTSET's effect on WT and C505T channels. (A) I/V relations from WT CNG channels, in inside-out patches, before (Control), during (MTSET + cGMP), and after (Recovery) application of 2.5 mM MTSET, in the presence of 500 Î¼M cGMP (open state). Recovery was measured â¼1 min after MTSET washout. Currents were elicited by 200-ms voltage steps from â100 to 100 mV (20-mV increments). Holding potential was 0 mV. Traces are averages of five trials. (B) MTSET had no irreversible effect on C505T channels in the open state. MTSET was applied for â¼2.5 min at â40 mV in the presence of 500 Î¼M cGMP. (C) MTSET irreversibly inhibited WT channels. I/V relations before (Control) and after (Recovery) application of 2.5 mM MTSET, in the absence of cGMP. (D) MTSET had no effect on C505T channels, in the closed state. MTSET was applied for â¼2.5 min at â40 mV in the absence of cGMP. In Fig. 2Fig. 3Fig. 4Fig. 5 and Fig. 7Fig. 8Fig. 9, the thick bar indicates the duration of MTS application and the thin bar indicates either the application of 500 Î¼M cGMP to the intracellular side of the membrane (for inside-out patches) or the time during which cGMP was present in the absence of Mg2+ (for outside-out patches). Unless otherwise indicated, membrane potential was always maintained at â40 mV.
	Figure 7. MTSET's effect on single T16C channels. (Top) Representative single channel traces before the application of MTSET (left) and corresponding amplitude histogram from 1 min continuous recording at â60 mV (right). (Bottom) Representative single channel traces after the application of MTSET to the inner side of membrane patches, in the absence of cGMP (left) and corresponding amplitude histogram from 1 min continuous recording at â60 mV (right). (c) Current level corresponding to the channel closed level.
	Figure 5. MTSET's effect on single-channel currents from W9C and L12C mutants. (A) W9C mutant single-channel traces before (top) and after (bottom) MTSET application to the inner side of the membrane patch, at â80 mV, in the presence of 1 mM cGMP. The amplitude histograms were obtained from â¼10 s of continuous recording and reveal that at least two active channels were present in the patch. After MTSET application, the channel activity decreased considerably. This patch is representative of five experiments. (B) L12C mutant single-channel currents before (top) and after (bottom) MTSET application to the inner side of the membrane patch, at â100 mV, in the presence of 1 mM cGMP. In L12C, the increased frequency of channel fluctuations did not allow us to resolve a clear peak corresponding to the open state. However, the inspection of the current traces and amplitude histograms did not reveal any major change in channel conductance, whereas the lobe corresponding to the open state in the amplitude histogram became more populated, suggesting that the channel open probability increased after treatment.
	Figure 3. MTSET effect on V4C and L7C mutant channels. (A) MTSET application on the inner side of membrane patches. (Top) MTSET's effect on V4C mutant in the closed (c.s.) and open (o.s.) state. (Bottom) MTSET's effect on L7C mutant in the closed (c.s.) and open (o.s.) state. (B) MTSET application on the outer side of membrane patches. (Top) MTSET's effect on V4C mutant in the closed (c.s.) and open (o.s.) state. (Bottom) MTSET's effect on L7C mutant in the closed (c.s.) and open (o.s.) state.
	Figure 6. MTSET effect on T16C channels. (A) MTSET's effect on the inner side of membrane patches, in the presence of cGMP. (B) MTSET's effect on the inner side of membrane patches, in the absence of cGMP. (c) MTSET's effect on the outer side of membrane patches, in the presence of cGMP. (D) I/V relations before and after the application of MTSET on the inner side of membrane patches, in the absence of cGMP. Membrane potential was stepped from â100 to +100 mV (20-mV increments). Holding potential was 0 mV. Currents are the difference between the patch current in the presence and absence of cGMP. Current traces were the average of five consecutive trials.
	Figure 13. Model of the proposed topology of two adjacent P loops in CNG channels. Arrows indicate the suggested displacements occurring during channel opening. Red circles represent proposed extracellular residues. Blue circles represent proposed intracellular residues. White circles represent residues not accessible to MTS compounds or residues which, when substituted with cysteine, prevented the expression of functional channels. Residues are numbered according to their position in the CNG channel protein (Kaupp et al. 1989). The residue position corresponding to the conventional numbering R1âS27 used in this paper is given within brackets. (Extra) Extracellular side, (Intra) intracellular side.
	Figure 8. MTSET's effect on I17C mutant. (A) I17C current decay in inside-out membrane patches during continuous recording at â40 mV. (Top) Current rundown in the absence of cGMP. Residual current was sampled every 30 s by brief application of cGMP (short bars). (Middle) Current rundown in the presence of cGMP. Baseline level was checked every 30 s by briefly applying solution without cGMP. (Bottom) Current t1/2 from 17 experiments in the absence (closed state) or presence (open state) of cGMP. Experimental procedure was as shown in top and middle panels. (B) I17C current decay in inside-out membrane patches during continuous recording at â40 mV in the presence of MTSET. (Top) Current rundown in the presence of cGMP. (Bottom) Current t1/2 from 20 experiments in the presence of cGMP and MTSET (Open state + MTSET) and of cGMP in the absence of MTSET (open state).
	Figure 9. MTSET effect on T20C and P22C mutant channels. (A) MTSET application on the inner side of membrane patches. (Top) MTSET effect on T20C mutant in the closed (c.s.) and open (o.s.) state. (Bottom) MTSET effect on P22C mutant in the closed (c.s.) and open (o.s.) state. (B) MTSET application on the outer side of membrane patches. (Top) MTSET's effect on T20C mutant in the closed (c.s.) and open (o.s.) state. (Bottom) MTSET's effect on P22C mutant in the closed (c.s.) and open (o.s.) state.
	Figure 10. Cysteine prevented MTSEA effect on T20C and P22C channels. (A) 10 mM cysteine applied to the outer side of membrane patches had no effect on T20C currents. (Left) Cysteine applied in the open state. (Right) Cysteine applied in the absence of cGMP. (B) Effect of MTSEA applied to the inner side of patches containing T20C channels in the open state (o.s.). (Left) MTSEA irreversibly inhibited T20C current. (Right) 10 mM cysteine on the outer side of the patch prevented the irreversible block produced by MTSEA applied to the patch inner side. (C) Effect of MTSEA applied to the inner side of patches containing T20C channels in the closed state (c.s.). (Left) MTSEA irreversibly inhibited T20C current. (Right) 10 mM cysteine on the outer side of the patch prevented the irreversible block produced by MTSEA applied to the patch inner side. (D) Effect of MTSEA applied to the inner side of patches containing P22C channels in the closed state (c.s.). (Left) MTSEA irreversibly inhibited P22C current. (Right) 10 mM cysteine on the outer side of the patch prevented the irreversible block produced by MTSEA applied to the patch inner side.
	Figure 11. MTSEA's effect on P22C single-channel currents. (Top) P22C single-channel traces before MTSEA application to the inner side of the membrane patch at â100 mV in the presence of 1 mM cGMP. (Bottom) Current recording after treatment with MTSEA. The amplitude histograms were obtained from â¼10 s of continuous recording and show that channel activity decreased drastically after MTSEA application. The amplitude histograms obtained after treatment were identical irrespective of whether cGMP was present or not, whereas cGMP application before treatment stimulated the typical intense ânoisyâ CNG channel activity reflected in the wide amplitude histogram, with a peak around â2.2 pA. This patch is representative of three experiments.
	Figure 12. Summary of MTSET effects on mutant channels on the indicated mutants. (A) Intracellular MTSET effect in the absence of cGMP. (B) Intracellular MTSET effect in the presence of cGMP. (C) Extracellular MTSET effect in the presence of cGMP. The experimental procedure was explained in Fig. 3. The dashed lines mark the 20% level of inhibition or potentiation, which we considered significant for inferences about residue accessibility (see text).

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