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Cyclic nucleotide-gated (CNG) channels are tetrameric non-specific cation channels. They mediate the receptor potentials in photoreceptors and cells of the olfactory epithelium and they are activated by the binding of cyclic nucleotides such as cGMP and cAMP. Previous studies in homotetrameric CNGA1 channels, activated with covalently bound cGMP, presented evidence that partially liganded channels cause partial channel opening (Ruiz & Karpen, 1997, 1999). Here, homotetrameric CNGA1 channels were expressed in Xenopus oocytes. Conductance and gating of these channels were studied as a function of the concentration of freely diffusible cGMP and with different permeating ions. At saturating cGMP the current levels distributed around a single mean in a Gaussian fashion and the open times were long. At low cGMP, however, the current levels were heterogeneous: they were smaller than those at saturating cGMP, equal, or larger. The open times were short. Ions generating the larger single-channel currents (Na+ > K+ > Rb+) concomitantly increased the heterogeneity of current levels and decreased the open probability and open times. The results suggest that the activation of CNGA1 channels by cGMP and ions staying longer in the pore is associated with less extensive and less frequent conformational fluctuations of the channel pore.
When studying basic functional properties of CNG channels, homomultimeric channels, expressed in appropriate heterologous cells, are often used because they comprise a defined composition. Gamel & Torre (2000) reported that Na+ and K+ ions modulate the channel gating in homomultimeric CNGA1 channels (for nomenclature see Bradley et al. 2001; Kaupp & Seifert, 2002). Recently, Holmgren (2003) studied the effect of permeating Na+ and K+ ions on the gating of CNGA1 channels at the single-channel level: at cGMP concentrations generating large open probability (Po), intracellular K+ caused higher Po than intracellular Na+ by prolonging the open channel lifetime. The author presented evidence that this effect is mediated by the interaction of the permeating ions with the pore. This interpretation is consistent with the idea that the selectivity filter of the CNG channel pore takes part in the gating (Sun et al. 1996; Bucossi et al. 1997; Becchetti et al. 1999; Liu & Siegelbaum, 2000; Flynn & Zagotta, 2001; Tränkner et al. 2004).
At cGMP concentrations generating low Po and with Na+ as permeating ion, native CNG channels produce sublevel openings (Hanke et al. 1988; Ildéfonse & Bennett, 1991; Taylor & Baylor, 1995). These results suggest that partial liganding at low cGMP promotes partial channel gating whereas full liganding at saturating cGMP promotes gating to the fully open channel. Further support for this hypothesis comes from results in homomultimeric CNGA1 channels locked in different ligand-bound states: with Na+ as permeating ion, two sublevels were observed and their amplitude was attributed to the number of cGMP molecules bound to the channel (Ruiz & Karpen, 1997, 1999). These authors also presented evidence that low concentrations of freely diffusible cGMP generate respective sublevels. In contrast, with K+ as permeating ion and freely diffusible cGMP the fully open level prevails at both saturating and low cGMP (Benndorf et al. 1999). These results show that the incidence of sublevels does not strictly depend on the cGMP concentration but also on the type of permeating ion. Hence, the permeating ions modulate Po not only by altering the lifetime in the fully open state but also by affecting the channel conductance.
In this study, we have examined systematically the influence of cGMP and permeating ions on CNGA1 channel gating and conductance. We show that saturating cGMP generates long openings with a single current level, whereas low cGMP generates short openings with multiple current levels, including sublevels, the level observed at saturating cGMP, and superlevels. Ions that stay in the pore longer (Rb+ > K+ > Na+) prolong the open times and decrease the multiplicity of current levels compared to ions staying in the pore for shorter times. The results suggest that at low cGMP the pore conformation fluctuates rapidly between multiple states of which some are open, whereas at saturating cGMP, the frequency and extent of the conformational fluctuations are strongly reduced. Permeating ions modulate these conformational fluctuations.
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In this study we reconsidered the gating of single homomultimeric CNGA1 channels as a function of the cGMP concentration and the type of permeating ion. Our results demonstrate that an increased cGMP concentration correlates not only with higher Po and longer open times but also with a decreased incidence of heterogeneous current levels. Ions staying longer in the pore affect these functional parameters similarly but the extent of these ionic effects is smaller than that of the cyclic nucleotide. Because the selectivity filter is the principal gate for the cyclic nucleotide-induced gating (Sun et al. 1996; Bucossi et al. 1997; Becchetti et al. 1999; Liu & Siegelbaum, 2000; Flynn & Zagotta, 2001) and the ions affect the gating when permeating the pore (Fig. 7; cf. Holmgren, 2003), all gating effects described in this paper can be attributed to conformational changes of the channel pore.
The pronounced heterogeneity of current levels at low cGMP (low Po) indicates that the pore conformation can adopt multiple open states. The individual events, however, contained mostly only a single level, even in multichannel patches. This result shows that the switching of the channel to the next conductance state must proceed when the channel pore is closed. It is therefore likely that the pore passes at least two closed states between two open states with different conductance. The fact that at low Po the sojourns of the pore in closed states are much longer than those in open states makes it likely that even more closed states are passed between two open states. It is therefore suggested that at low Po the pore conformation fluctuates between multiple states of which only a small fraction is open. The conductance of these open states can be different, generating the heterogeneity of current levels.
At saturating cGMP, the absence of heterogeneous current levels indicates that the high concentrations of the bound cyclic nucleotide prevents the pore from adopting most of the open pore conformations in the sense that the pore is ‘frozen’ in conformations generating only one conductance. Because of the biexponential distributions of the open times, there are at least two open pore conformations. The effect of increasing the cGMP concentration on the pore can therefore be understood as a reduction of the number of accessible states. The high Po suggests that the accessibility of closed states is significantly more reduced than that of the open states. A cGMP-induced reduction of the number of accessible closed and open states can also explain the longer open times at high than at low cGMP: with fewer accessible states at high cGMP, fewer transitions are possible along which an open state can be left. Hence, increasing cGMP does not only reduce the extent of conformational pore fluctuations, as suggested by a reduced number of current levels, but also the frequency of these fluctuations, as suggested by the longer open times.
The effect of the permeating ions on the gating is also directed on the conformational fluctuations of the pore; ions staying longer in the pore (Rb+ > K+ > Na+) exert similar effects to an increased cGMP concentration; at low cGMP the number of accessible conductance states is reduced, Po and the open times are increased. The effects of ions, however, are only weak compared to those of the cGMP concentration. The result that at saturating cGMP the heterogeneity of levels is lost with all ion species supports the notion that ions and cGMP control the extent of conformational fluctuations via the same mechanism. Despite this result, our data also suggest that ions and cGMP affect different closing reactions because the open times at saturating cGMP remain different with the different ions (Fig. 7D). This result supports the notion that ions and cGMP control the frequency of conformational fluctuations by involving different closing reactions. Evidence for different closing reactions affected by ions and cGMP was also derived above from the analysis of Po and the open times (see text to Fig. 6).
We observed that the different amplitudes of the Rb+, K+ and Na+ currents at saturating cGMP negatively correlate with the degree of heterogeneity of current levels and the prolongation of open times. From a mechanistic point of view it is likely that these effects of ions depend on the dwell time of the ions in the pore. With single-channel currents in the range of 0.5 pA (Rb+) to 3.6 pA (Na+), the upper limit of the mean dwell time of the ion in the channel is 3.2 × 10−7 to 4.5 × 10−8 s. Since these dwell times are much shorter than the open times, an ion binding in the pore cannot stabilize the open channel directly by its dwell time at the binding site. One possible explanation is that several ions bind in the pore simultaneously and that channel closure is only possible when both (or all) binding sites are empty. Evidence for the simultaneous binding of two ions in the selectivity filter has been provided for K+ channels (Doyle et al. 1998). Alternatively, it is conceivable that the mean time an ion occupies a single binding site is transformed to decreased conformational fluctuations of the pore region.
Gamel & Torre (2000) were the first to show that permeating ions modulate the gating of CNGA1 currents. Replacing Na+ on the inside by K+ slowed down the channel gating at positive voltages. This result fits with the idea derived from this study that K+ ions reduce the frequency of conformational fluctuations with respect to Na+ ions. Our results also confirm the previous results of Holmgren (2003) who observed at cGMP concentrations generating large Po that permeating K+ ions prolong the open-channel lifetime with respect to permeating Na+ ions.
In previous work on the incidence of sublevel openings at low cGMP, Ruiz & Karpen (1997, 1999) reported that CNGA1 channels locked in partially liganded states with 8-p-azidophenacylthio- cGMP (APT-cGMP) produce distinct sublevels and the authors identified these sublevels also when activating the channels with free cGMP. With symmetrical Na+ and at low cGMP we also observed a preference for sublevel openings but our amplitude histograms did not confirm the notion that partial liganding causes only partial opening (Figs 3 and 4). Generally, when relating results obtained with permanently bound APT-cGMP to those obtained with freely binding cGMP, one should be aware that this is only possible if the binding/unbinding reaction of the free cGMP is slow compared to the subsequent allosteric reaction. This has not been shown so far. If the allosteric reaction is slower than the binding reaction then the observed amplitude heterogeneity in our measurements (including the superlevels) could be lost in the experiments of Ruiz & Karpen simply by the permanent binding of the APT-cGMP. Hence, our results do not necessarily conflict with those of Ruiz & Karpen (1997, 1999) obtained with permanently bound APT-cGMP but reflect the channel action with freely diffusible cGMP.
The present data also explain contradictory results on the incidence of sublevels at partially activated CNGA1 channels in the studies of Ruiz & Karpen (1997, 1999) and our work (Benndorf et al. 1999): while Ruiz & Karpen (1999) reported that sublevel openings dominate the channel activity, we observed a mean current level close to the level observed at saturating cGMP (Benndorf et al. 1999). The results of this study show that permeating Na+ ions particularly promote sublevels whereas permeating K+ ions promote levels both smaller and larger than the level observed at saturating cGMP, resulting in amplitude histograms with an open-level distribution apparently close to that for the level observed at saturating cGMP. The fact that at very low Po (1.3 and 10 μm cGMP; Fig. 6B) the channels generated not only sublevels but also the level observed at saturating cGMP and superlevels, rules out that a partially liganded channel produces a distinct subconductance state.
Native channels are heterotetramers composed of three CNGA1 subunits and one modulating CNGB1 subunit (Zheng et al. 2002; Weitz et al. 2002; Zhong et al. 2002). Two of the modulating effects of the CNGB1 subunit are to decrease the single-channel conductance and to increase the open-channel noise, if compared to homotetrameric CNGA1 channels (Körschen et al. 1995). In the context of the present study these results suggest that the CNGB1 subunit also modulates the conformational fluctuations of the channels. In future experiments it would be of interest to examine how this modulation is related to the effects of cGMP and the ions described here. Other interesting areas for further study are conformational fluctuations at physiological voltages and with physiological ions, including external Ca2+ ions. Ca2+ ions can be expected to conspicuously decrease conformational fluctuations because they permeate the pore only slowly (cf. Frings et al. 1995).