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    • Cinacalcet synthesis The ion conduction pathway reported her

    2022-01-17

    The ion conduction pathway reported herein accounts for all known functional properties of EAAT/GltPh anion channels. Simulations reveal unitary current amplitudes and ion selectivities (Figures 3C and 3D) that resemble experimental results (Melzer et al., 2003, Wadiche and Kavanaugh, 1998). The calculated minimum pore diameter of GltPh is ∼5.6 Å (Figure 3E), which perfectly fits the predicted minimum pore diameter of >5 Å based on anion substitution experiments on EAAT1 anion Cinacalcet synthesis (Wadiche and Kavanaugh, 1998). Rapid substrate application experiments have shown that EAAT anion channel activation is delayed compared with glutamate translocation (Grewer et al., 2000). These findings indicate that anion-conducting states existing “outside” the glutamate uptake cycle can be explained by channel opening as a branching reaction from ICs (Figure 1). Simulations predict voltage independence of anion channel opening within the physiological voltage range (Figure 2B). This result explains the experimental observation that the voltage- and substrate dependence of EAAT anion channels are tightly linked to transitions within the transport cycle (Bergles et al., 2002, Machtens et al., 2011a). Simulated anion permeation is unchanged in both the presence and absence of bound substrate (Figure 3E), as expected from the experimental unitary conductances being indistinguishable in the presence and absence of glutamate (Kovermann et al., 2010). Because anion channel opening is tightly linked to translocation of the transport domain, our results indicate that transport substrates increase EAAT anion currents by promoting intermediate states. Distinct EAAT isoforms differ strongly in the relative amplitudes of their transport and anion currents (Fairman et al., 1995, Mim et al., 2005). However, analysis of unitary current amplitudes revealed similar single-channel amplitudes (Schneider et al., 2014, Torres-Salazar and Fahlke, 2007). The high degree of conservation of pore-forming residues (Figure S4) is consistent with the similarities in anion channel unitary current amplitudes and selectivity of different transporter isoforms. Lastly, the novel anion conducting conformation can account for all published mutagenesis and crosslinking results on EAAT anion conduction (Ryan et al., 2004, Shabaneh et al., 2014). The “S65 path” (Figure S7) is the only location of the anion channel that has been discussed in recent years. We could not find any indication for a direct contribution of this region to anion permeation. Our simulations show that the “S65 path” is hydrated in ChC, thereby suggesting that S65 and adjacent residues could be involved in facilitating the opening of the transport/trimerization domain interface instead. We thus speculate that the “S65 path” may modulate formation of the ChC conformation, which provides an explanation for the impact of mutations in this region on anion channel function (Cater et al., 2014, Ryan and Mindell, 2007, Ryan et al., 2004). The positive electrostatic potential necessary for perfect anion selectivity of EAAT/GltPh anion channels is provided by a single positively charged side chain, R276. Surprisingly, during evolution, this arginine has moved from the tip of HP1 in GltPh to TM8 in EAATs, while retaining a similar side chain position in the tertiary structure. In GltPh, as well as in EAATs, this arginine has been implicated in binding amino acid substrates, as well as binding Na+ and K+ (Ryan et al., 2010, Verdon et al., 2014). Unitary anion conductance is not affected by aspartate (Figure 3), indicating that the interaction of R276 with transport substrates does not modify its effect on anion conduction and selectivity. The tight linkage between anion channel gating and glutamate transport in EAAT/GltPh was previously explained by assuming that certain states of the transport cycle are anion conducting (Bergles et al., 2002). Because GltPh structures did not exhibit an open pore with dimensions that might account for the experimentally observed anion conduction properties, it was recently suggested that additional yet to be defined ICs that occur during translocation might be anion conducting (Cater et al., 2014). We have now demonstrated that intermediate transport conformations are nonconducting and that EAAT/GltPh anion channel opening transitions require the lateral movement of the glutamate transport domain together with pore hydration from intermediates. Anion channel opening is therefore not part of the transport cycle, but instead is achieved via a branching conformational change. This design permits rapid transition through the full transport cycle without anion channel opening. Furthermore, it allows certain EAAT isoforms to function as effective transporters, with low anion channel open probabilities, and other isoforms to have low transport rates but high occupations of the anion channel mode.