Antonio Peres
Department of Biotechnology and Molecular Sciences, University of Insubria, Varese, Italy

https://doi.org/10.36866/pn.70.18

The problem of coupling high selectivity with high flux rate in ionic channels has been clarified by Roderick MacKinnon, who received the Nobel prize in 2003 for his studies on the potassium channel KcsA (MacKinnon, 2003). Fig. 1A illustrates the structure of the selectivity filter of KcsA, formed by the ‘potassium signature’ residues TTVGYG. Each of the four subunits that constitute the oligomeric assembly contributes four main-chain carbonyl atoms that surrogate the interactions between K+ ions and water in the bulk solution. The resulting organisation of the K+ binding sites in the pore, in addition to providing selectivity by way of its intrinsic electrostatic properties, also allows high throughput, because the proximity of the potassium sites causes the ions to hop from one place to the next due to charge repulsion.

This elegant demonstration confirms an explanation proposed many years before, on the basis of purely functional observations: that of selectivity by affinity (Hess & Tsien, 1984). This idea developed from results on Ca2+-selective channels: how was it possible for a channel to choose Ca2+ over Na+, an ion with similar dimensions but much more concentrated in the extracellular medium? The observation that when Ca2+ was absent Na+ ions were able to generate a much larger current through the same channels led to the notion of a ‘sticky’ channel, i.e. to the idea that the pore contained a high-affinity binding site for Ca2+.

This was later identified in a ring of four glutamates (the EEEE motif), each located in the same relative position of the P-loops of the four homologous domains of the protein. Structural modelling of this region (Lipkind & Fozzard, 2001) showed that a Ca2+ ion is strongly bound by the carboxyl oxygens of the glutamates (Fig. 1B). At physiological Ca2+ concentrations, however, this electrostatic trap is weakened by the simultaneous presence of two other Ca2+ ions bound to two flanking low-affinity sites, allowing a significant flux to occur.

Thus, K+ channels and Ca2+ channels appear to teach us that efficient discrimination among similar ion species requires a toll in terms of a significant reduction in conductivity, and that further structural specialisations are needed to increase flux rate.

Selectivity in transporters

The application of electro-physiological and biophysical approaches to the study of electrogenic cotransporters is a relatively young area of research, that has become possible following the cloning of the proteins of interest and their overexpression in heterologous systems. This delayed start has offered in return the advantage of finding ready-to-use ideas and rationales that had been previously developed in the field of ionic channels. Many analogies between channels and transporters have been found, such as channel-like behaviour of transporters, transient currents reminiscent of the gating currents of voltage-dependent channels, and properties compatible with single-file permeation mechanisms.

The observations described in our recent paper (Miszner et al. 2007) point to a further possible analogy between transporters and channels, namely that selectivity by affinity may constitute a useful idea also in understanding the functioning of transporters.

Indeed, the competition experiments reported by Miszner et al. strongly recall the Ca2+ block of the current carried by other ion species in the Ca2+ channel (Hess & Tsien, 1984). The neutral amino acid transporter KAAT1, cloned from the midgut of the larva of the invertebrate Manduca sexta, has a rather wide spectrum of transport-able substrates and, in addition, is able to exploit either the Na+ or the K+ electrochemical gradients as energy sources for active transport. In this transporter leucine acts as a dominant substrate: although in presence of Na+ threonine and proline may be transported at higher rates, the concomitant presence of leucine, even at a lower concentration, reduces the amplitude of the transport-associated current to the level produced by leucine alone (Fig. 2A).

This electrophysiological observation is confirmed by radioactive uptake experiments, that show that in presence of both leucine and threonine, or leucine and proline, only leucine is actually translocated. Quite interestingly, the negative dominance seen in presence of Na+, is reversed in presence of K+: in this condition the leucine-associated current is higher, and addition of leucine increases the amplitude of the currents elicited by threonine or proline alone (Fig. 2B). This last finding represents a variant with respect to the paradigm relating high selectivity and low flux rate, and will require better knowledge of the substrate-transporter interactions and of the conformational changes involved in the substrate translocation.
KAAT1 belongs to the SLC6A family of transporters, a bacterial member of which, LeuTAa, has been recently crystallized and its atomic structure resolved (Yamashita et al. 2005). On the basis of the leucine binding site of LeuTAa, and examining the differences in sequence between KAAT1 and CAATCH1 (a very similar transporter in which leucine act as a blocker), we have identified serine 308 in KAAT1 (S256 in LeuTAa and T308 in CAATCH1) as a critical residue for leucine transport. Replacing S308 in KAAT1 with the threonine present in CAATCH1 converts leucine from a transported substrate to a blocker. Indeed in a transport system whose selectivity is based on high-affinity binding sites, the difference between a permeating species and a blocker may be rather subtle, as shown by Ca2+ ions in Ca2+ channels (Hess & Tsien, 1984). The S308T mutation in KAAT1 may simply increase the strength of leucine binding to such an extent that translocation or dissociation very rarely occur, thereby blocking the transporter.

The amino acid corresponding to KAAT1 S308 has been found to play an important role in substrate selectivity in other transporters of the family as well. The two glycine transporters GlyT1b and GlyT2a may be distinguished by their differential ability, shown by the former but not by the latter, to transport also sarcosine. Replacing serine 481 of GlyT2a (corresponding to S308 of KAAT1) with the glycine present in GlyT1b makes GlyT2a capable of sarcosine transport, while the reverse mutation G305S in GlyT1b suppresses sarcosine transport (Vandenberg et al. 2007). Furthermore, the creatine transporter CRT may be induced to transport GABA by a number of selected amino acid substitutions, among which the replacement of glycine 318 (corresponding to S308 of KAAT1) with the alanine present in the same position in the GABA transporter GAT1 (Dodd & Christie, 2007).

It appears, therefore, that position 256 of LeuTAa, located in the unwound region of the sixth transmembrane segment, plays a fundamental – though not exclusive – role in determining substrate specificity in a number of SLC6A transporters.

In ionic channels selectivity and permeation involve only two chemical species, the ion and the protein. Conversely, in cotransporters at least three actors are involved: the protein, the ion(s) and the organic substrate. This is very clearly demonstrated in the snapshot of the structure of LeuTAa
(Fig. 1C), in which a Na+ ion is shown to participate in shaping the leucine binding pocket. Furthermore, the controversial role of Cl- ions in this family of transporters is beginning to be elucidated as an optional fourth component, in those transporters of this family that lack an acidic residue in the position corresponding to E290 of LeuTAa (Forrest et al. 2007; Zomot et al. 2007).

Indeed, as already noted (Kilic & Rudnick, 2000), whereas ions present a symmetrical surface to the pore walls of the channel, the small organic molecules that are translocated by cotransporters are generally highly asymmetric and would consequently require an asymmetrical binding site. It is not a surprise then that while the pore of the K+ and Ca2+ channels is at the centre of a tetrameric assembly, each subunit of the oligomeric transporters appears to function independently (Yamashita et al. 2005; Boudker et al. 2007). The lack of symmetry in transporters implies greater complexities in the number and types of interactions among the distinct molecular entities involved. The recent definition of the atomic structure of LeuTAa has offered the tools for the study of these interactions, and the challenge is now to understand their dynamics during the transport cycle.

Acknowledgement

Many thanks are due to Dino Fesce, Elena Bossi and Stefano Giovannardi for critical reading of the manuscript.

References

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