Modificaciones posteriores de los fondos de pensiones

The biggest question with repect to their physiological function is still whether delta sub- units serve as ion channels in vivo. In this regard, arguments for a mere metabotropic function of delta2 receptors stem from two lines of evidence: First, an early pharmacolog- ical study had shown that AMPA receptor antagonists can block EPSCs at the parallel ber Purkinje cell synapse in the cerebellum (Kano and Kato, 1987). Furthermore, the amplitudes and kinetics of mEPSCs are normal in delta2 knock out mice (Jeromin et al., 1996; Yawata et al., 2006). Both of these ndings have led to the conclusion that delta2 receptors do not contribute to EPSCs at the parallel ber Purkinje cell synapse.

The second line of evidence stems from transgenic rescue studies showing that the disruption of delta2 ligand binding or ion permeability does not interfere with the mutant subunit's ability to rescue the abrogated synaptic plasticity in delta2-decient mice (Hirai et al., 2005; Yuzaki, 2005; Kakegawa et al., 2007b,a). Hence, ion channel function seems not to be required for the subunit's involvement in cerebellar synaptic plasticity.

On the other hand, the spontaneous current through homomeric delta2-lurcher chan- nels clearly showed that the receptor is capable of uxing ions, although in this case only

passively and not ligand-induced. Two ndings showed, however, that the LBD of delta2 does communicate its status to the ion pore: Both D-serine and an antibody directed against the LBD of delta2 reduced spontaneous current through delta2-lurcher channels (Naur et al., 2007; Hirai et al., 2003).

Assuming that the introduction of the GluR6 LBD into delta2 did not alter the general structure or the gating mechanism of that subunit, the presented ndings go one step further. They show that ligand-induced movement can gate delta2 channels that do not contain the lurcher mutation. This strongly suggests that the wild type receptors are capable of forming functional ion channels. The question remains under which conditions, and if these conditions play a physiological role.

A low anity D-serine receptor? Similar to the NR1 and NR3 subunits of NMDA receptors, it has been shown that the delta2-lurcher mutant is responsive to D-serine and glycine (Naur et al., 2007). However, D-serine application does not activate the wild type receptor, and the spontaneous current through delta2-lurcher is blocked by D-serine rather than D-serine causing additional current. One fundamental dierence of delta2 compared to NR1 is the much lower anity for D-serine and glycine, which is three orders of magnitude lower for the delta2 subunit (Naur et al., 2007). If delta receptors were activated by D-serine or glycine in vivo, comparably high concentrations would be needed to activate the receptors.

With regard to the availability of D-serine in suciently high concentration, two nd- ings are particularly interesting: First, D-serine concentrations are high in the developing cerebellum and restricted to Bergmann glia cells which ensheath parallel ber Purkinje cell synapses (Hashimoto et al., 1995; Schell et al., 1997). Concentrations peak at P14 and then decline to negligible levels in the adult. Second, the eective concentration necessary to reduce spontaneous current through delta2-lurcher channels varies with extracellular calcium. D-serine is more potent in calcium-free solutions (∼300 µM at 0 mM calcium), while at physiological calcium concentrations the potency declines to values in the mil- limolar range (∼3 mM at 3 mM calcium) (Hansen et al., 2007b). Considering that the potency of D-serine was determined for the delta2-lurcher mutant (which decisively in- creases agonist potency in other iGluRs), these ndings suggests that in vivo the D-serine levels might not be sucient to aect delta2, at least not in the adult.

A sensor for extracellular calcium depletion? What if extracellular calcium were the decisive signal for delta receptor function? That extracellular calcium can be a rst messenger is not unprecedented. The human Ca2+_{-sensing receptor is a G protein-coupled}

receptor that is activated by rising extracellular calcium. Most interestingly, it belongs to the same family of GPCRs as the mGluRs, and its ectodomain is related to bacterial periplasmic binding proteins (Brown et al., 1993; Hu and Spiegel, 2007).

It is not intuitively obvious, however, that calcium might serve as an important rst messenger. The ion occurs in at least 10000-fold higher concentration extracellularly than in the intracellular space. Nevertheless, there is accumulating evidence that locally con- ned changes of extracellular calcium are sensed by cells and have profound consequences for synaptic transmission (Brown et al., 1995; Rusakov, 2001; Rusakov and Fine, 2003). Simulations indicate that substantial depletions (1.5 to 0.8 mM) of extracellular calcium in the synaptic cleft can occur during high frequency stimulations (Vassilev et al., 1997). The delta receptors might therefore be involved in sensing a depletion in extracellular calcium. If and how such a signal might be transduced to pore opening remains to be elucidated. Since calcium apparently binds to the dimer interface of two adjacent delta2 subunits and stabilizes it, the mechanism of transducing a reduction in extracellular cal- cium would have to be distinct from the 'normal' cleft closure mechanism induced by glutamate at conventional iGluRs. Such a fundamental dierence in gating and activa- tion would not just t nicely with the described observation that a 'normal' LBD can gate delta2, but could also be reconciled with the fact that the receptors apparently do not contribute to EPSCs. They might be activated only under dened conditions of ex- tracellular pH and calcium concentrations, and on top of that be dependent on D-serine availability and thus be developmentally regulated.

Ionotropic glutamate receptors (iGluRs) critically shape excitatory synaptic signals in the central nervous system. Contrary to the well-characterized AMPA, kainate, and NMDA receptor subtypes, no agonist-induced ionotropic function of the two wild type delta re- ceptor subunits, delta1 and delta2, has yet been described. Based on sequence similarity, the delta subunits clearly comprise a separate subtype of iGluR. However, their non- responsiveness to glutamatergic agonists earned them the designation 'orphan' receptors. Even now that the delta2 subunit has recently been relieved from its 'orphan' receptor status by the elucidation of its ligand binding domain (LBD) structure, the wild type receptor remains unresponsive to molecules that arguably bind to its LBD. As a conse- quence, electrophysiological characterization of the delta subunits remains limited to the delta2-lurcher point mutant which spontaneously gates in the absence of ligand. Since the introduction of the lurcher mutation does not confer spontaneous gating to delta1, no electrophysiological data is available on this subunit, making it the least examined mammalian iGluR subunit.

To investigate the capability of single delta receptor domains to support ionotropic function, those domains that decisively govern iGluR properties, namely the ion pore, the LBD, and the linkers that connect the two, were examined by reciprocal domain transplan- tation. The domains were exchanged between the delta subunits and the AMPA receptor subunit GluR1 or the kainate receptor subunit GluR6. Electrophysiological properties of the resulting chimeras were examined in Xenopus laevis oocytes and HEK293 cells.

The most profound inuence on channel function was observed upon transplantation of the three linkers, emphasizing the crucial role of these regions in glutamate receptor function. The linker transplantations showed that delta1- or delta2-derived linkers can support channel gating in GluR1 receptors, while GluR1 receptor linkers did not restore ionotropic function to delta2 receptors. This suggests that the linkers of delta receptors and GluR1 function similarly.

Interestingly, all three linkers decisively aected receptor functionality, agonist po- tency, and desensitization. The GluR1 chimera that carried the delta2-derived linker pre- ceding the rst transmembrane domain (TMD A) had properties virtually identical to the GluR1-lurcher mutant. Combining this new lurcher-like linker chimera with the original GluR1-lurcher mutation allowed the reassessment of the eect of lurcher on GluR1 gating properties. The observed dierential but interdependent inuence of linker and lurcher

mutations on receptor properties suggests that the linkers are part of a ne-tuned struc- tural element that normally stabilizes the closed ion pore. Lurcher-like mutations pro- posedly act by disrupting this element such that agonist-induced conformational changes are not necessarily required to gate the channel. Since all electrophysiological data on delta2 so far was obtained using delta2-lurcher, the mechanism of action of this mutation on AMPA receptors impacts the interpretation of the delta2 data.

The minimum requirement to induce key lurcher-like gating features proved to be the mutation of the ve amino acids N-terminally adjacent to TMD A. However, numerous mutations in this receptor region showed that the full lurcher eect apparently is cumu- lative and involves amino acid changes that spread over the entire linker. Unexpectedly, a leucine-to-isoleucine mutation at the position directly preceding TMD A substantially increased current amplitudes both in GluR1 and GluR6, suggesting that this site might serve some important, conserved function in glutamate receptor gating.

For delta1, ion pore and LBD transplantations yielded inconclusive results. Both a GluR2 receptor containing the ion pore of delta1 and the delta1 receptor containing the LBD of GluR2 showed clear responses in one experiment that could not yet be reliably reproduced. Nevertheless, these recordings represent the rst indication that delta1 chan- nels might actually support ion channel function.

Transplantations of the ion pore of delta2 were unsuccessful in creating functional chimeric receptors. However, the delta2 subunit formed functional glutamate-gated ion channels when its LBD was exchanged for that of GluR6. As opposed to all previous electrophysiological characterization attempts that analyzed spontaneous current through delta2-lurcher, this chimera allowed to characterize agonist-controlled current through a delta2 ion channel. The chimera proved to be eciently gated by agonists that activate GluR6, less inwardly rectifying than GluR1 and GluR6 Q variants, blocked by extracel- lularly applied naphthylacetyl spermine, and susceptible to lectin modulation. Although the delta2 pore features a glutamine at the position corresponding to the Q/R/N site in iGluRs, it was less calcium-permeable than the GluR6 Q variant. Interestingly, current amplitudes were reduced by lowering the intracellular calcium concentration, indicating that intracellular calcium might be a regulatory factor of delta2 channel properties.

In view of the nding that the LBD of delta2 binds glycine and D-serine and is deci- sively modulated by extracellular calcium, the present ndings strongly suggest that delta receptors can form functional ion channels, that the key to their role in synaptic plasticity is likely to be found within their LBD, and that both intra- and extracellular calcium play a decisive role in regulating their function.

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