Dynamic changes in the TRPA1 selectivity filter lead to progressive but reversible pore dilation.

TRPA1 is a nonselective cation channel belonging to the transient receptor potential (TRP) family that is expressed in peripheral sensory neurons and may play important roles in pain perception and inflammation. We found that agonist stimulation of TRPA1, along with other members of the TRP family (TRPV1-4 and TRPM8), can induce the appearance of a large pore permeable to large organic cations such as Yo-Pro (YP) and N-methyl-d-glucamine, in an agonist and divalent cation-dependent manner. YP uptake was not inhibited by a panel of putative gap junction/pannexin blockers, suggesting that gap junction proteins are not required in this process. Our data suggest that changes in the TRP channel selectivity filter itself result in a progressive but reversible pore dilation process, a process that is under strong regulation by external calcium ions. Our data suggest that calcium plays a novel role in setting the amount of time TRPA1 channels spend in a dilated state providing a mechanism that may limit sensory neuron activation by painful or irritating substances.

Open probability of homomeric murine 5-HT3A serotonin receptors depends on subunit occupancy.

1. The time course of macroscopic current responses of homomeric murine serotonin 5-HT3A receptors was studied in whole cells and excised membrane patches under voltage clamp in response to rapid application of serotonin. 2. Serotonin activated whole cell currents with an EC(50) value for the peak response of 2 microM and a Hill slope of 3.0 (n = 12), suggesting that the binding of at least three agonist molecules is required to open the channel. 3. Homomeric 5-HT3A receptors in excised membrane patches had a slow activation time course (mean +/- S.E.M. 10-90 % rise time 12.5 +/- 1.6 ms; n = 9 patches) for 100 microM serotonin. The apparent activation rate was estimated by fitting an exponential function to the rising phase of responses to supramaximal serotonin to be 136 s(-1). 4. The 5-HT3A receptor response to 100 microM serotonin in outside-out patches (n = 19) and whole cells (n = 41) desensitized with a variable rate that accelerated throughout the experiment. The time course for desensitization was described by two exponential components (for patches tau(slow) 1006 +/- 139 ms, amplitude 31 %; tau(fast) 176 +/- 25 ms, amplitude 69 %). 5. Deactivation of the response following serotonin removal from excised membrane patches (n = 8) and whole cells (n = 29) was described by a dual exponential time course with time constants similar to those for desensitization (for patches tau(slow) 838 +/- 217 ms, 55 % amplitude; tau(fast) 213 +/- 44 ms, 45 % amplitude). 6. In most patches (6 of 8), the deactivation time course in response to a brief 1-5 ms pulse of serotonin was similar to or slower than desensitization. This suggests that the continued presence of agonist can induce desensitization with a similar or more rapid time course than agonist unbinding. The difference between the time course for deactivation and desensitization was voltage independent over the range -100 to -40 mV in patches (n = 4) and -100 to +50 mV in whole cells (n = 4), suggesting desensitization of these receptors in the presence of serotonin does not reflect a voltage-dependent block of the channel by agonist. 7. Simultaneously fitting the macroscopic 5-HT3A receptor responses in patches to submaximal (2 microM) and maximal (100 microM) concentrations of serotonin to a variety of state models suggests that homomeric 5-HT3A receptors require the binding of three agonists to open and possess a peak open probability greater than 0.8. Our modelling also suggests that channel open probability varies with the number of serotonin molecules bound to the receptor, with a reduced open probability for fully liganded receptors. Increasing the desensitization rate constants in this model can generate desensitization that is more rapid than deactivation, as observed in a subpopulation of our patches.

Identification of amino acid residues in GluR1 responsible for ligand binding and desensitization.

Although GluR1(o) and GluR3(o) are homologous at the amino acid level, GluR3(o) desensitizes approximately threefold faster than GluR1(o). By creating chimeras of GluR1(o) and GluR3(o) and point amino acid exchanges in their S2 regions, two residues were identified to be critical for GluR1(o) desensitization: Y716 and the R/G RNA-edited site, R757. With creation of the double-point mutant (Y716F, R757G)GluR1(o), complete exchange of the desensitization rate of GluR1(o) to that of GluR3(o) was obtained. In addition, both the potency and affinity of the subtype-selective agonist bromohomoibotenic acid were exchanged by the Y716F mutation. A model is proposed of the AMPA receptor binding site whereby a hydrogen-bonding matrix of water molecules plays an important role in determining both ligand affinity and receptor desensitization properties. Residues Y716 in GluR1 and F728 in GluR3 differentially interact with this matrix to affect the binding affinity of some ligands, providing the possibility of developing subtype-selective compounds.

Agonist discrimination between AMPA receptor subtypes.

The lack of subtype-selective compounds for AMPA receptors (AMPA-R) led us to search for compounds with such selectivity. Homoibotenic acid analogues were investigated at recombinant GluR1o, GluR2o(R), GluR3o and GluR1o + 3o receptors expressed in Sf9 insect cells and affinities determined in [3H]AMPA radioligand binding experiments. (S)-4-bromohomoibotenic acid (BrHIBO) exhibited a 126-fold selectivity for GluR1o compared to GluR3o. Xenopus laevis oocytes were used to express functional homomeric and heteromeric recombinant AMPA-R and to determine BrHIBO potency (EC50) at these channels. (R,S)-BrHIBO exhibited a 37-fold selectivity range amongst the AMPA-R. It is hoped that BrHIBO can be used as a lead structure for the development of other subtype-selective compounds.

Control of GluR1 AMPA receptor function by cAMP-dependent protein kinase.

Modulation of postsynaptic AMPA receptors in the brain by phosphorylation may play a role in the expression of synaptic plasticity at central excitatory synapses. It is known from biochemical studies that GluR1 AMPA receptor subunits can be phosphorylated within their C terminal by cAMP-dependent protein kinase A (PKA), which is colocalized with the phosphatase calcineurin (i.e., phosphatase 2B). We have examined the effect of PKA and calcineurin on the time course, peak open probability (P(O, PEAK)), and single-channel properties of glutamateevoked responses for neuronal AMPA receptors and homomeric GluR1(flip) receptors recorded in outside-out patches. Inclusion of purified catalytic subunit Calpha-PKA in the pipette solution increased neuronal AMPA receptor P(O,PEAK) (0.92) compared with recordings made with calcineurin included in the pipette (P(O,PEAK) 0.39). Similarly, Calpha-PKA increased P(O,PEAK) for recombinant GluR1 receptors (0. 78) compared with patches excised from cells cotransfected with a cDNA encoding the PKA peptide inhibitor PKI (P(O,PEAK) 0.50) or patches with calcineurin included in the pipette (P(O,PEAK) 0.42). Neither PKA nor calcineurin altered the amplitude of single-channel subconductance levels, weighted mean unitary current, mean channel open period, burst length, or macroscopic response waveform for recombinant GluR1 receptors. Substitution of an amino acid at the PKA phosphorylation site (S845A) on GluR1 eliminated the PKA-induced increase in P(O,PEAK), whereas the mutation of a Ca(2+), calmodulin-dependent kinase II and PKC phosphorylation site (S831A) was without effect. These results suggest that AMPA receptor peak response open probability can be increased by PKA through phosphorylation of GluR1 Ser845.

Novel potent AMPA analogues differentially affect desensitisation of AMPA receptors in cultured hippocampal neurons.

The agonist actions of two AMPA receptor analogues, (RS)-2-amino-3-(3-carboxy-5-methyl-4-isoxazolyl)propionic acid (ACPA) and (RS)-2-amino-3-(3-hydroxy-5-trfluoromethyl-4-isoxazolyl)prop ionic acid (Tri-F-AMPA) have been studied on cultured rat hippocampal neurons. Whole-cell recordings with semi-rapid application of the agonists were used to study steady-state (plateau) responses. ACPA was the most potent agonist (EC50, 1.2 microM), followed by AMPA (4.3 microM) and Tri-F-AMPA (4.6 microM), corresponding to a potency ratio of 4:1:1. Hill coefficients were close to 1 for AMPA and ACPA and close to 2 for Tri-F-AMPA, respectively. Plateau responses to maximal concentrations of the three agonists varied more than 2-fold. ACPA responses were 2.1 times greater and responses to Tri-F-AMPA were 1.6 times greater than responses to AMPA, respectively. Peak responses and desensitization were studied by using a fast piezoelectric device to apply agonists rapidly to outside-out patches. The time constants of desensitization were 8 ms for AMPA, 12 ms for Tri-F-AMPA and 17 ms for ACPA. There were no significant differences in the time-to-peak and 10-90% rise-time of the responses. The results indicate that of the three agonists tested, ACPA is the most potent at AMPA receptors expressed in cultured hippocampal neurons and that the maximum response to the agonists is inversely related to the rate of desensitization.

Pharmacological properties of homomeric and heteromeric GluR1o and GluR3o receptors.

Homomeric and heteromeric alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptor subunits GluR1o and GluR3o were expressed in Spodoptera frugiperda (Sf9) insect cells. Membranes containing the recombinant receptors showed a doublet of bands of the expected size (99-109 kDa) after western immunoblotting which was shifted to a single band upon deglycosylation. In (R,S)-[3H]AMPA binding experiments, high expression was seen (Bmax = 0.8-3.8 pmol/mg protein) along with high affinity binding to a single site (Kd, nM+/-S.D.): GluR1o, 32.5+/-2.7; GluR3o, 23.7+/-2.4; GluR1o + GluR3o, 18.1+/-2.9. The pharmacological profiles of these receptors resembled that of native rat brain AMPA receptors: AMPA analogues > L-glutamate > quinoxaline-2,3-diones > kainate. In the Xenopus oocyte expression system we had previously shown that the agonist (R,S)-2-amino-3-(3-carboxy-5-methyl-4-isoxazolyl)propionate (ACPA) exhibited an 11-fold selectivity for GluR3o vs. GluR1o. In this study, it was found that ACPA has 3-fold higher affinity at homomeric GluR3o and heteromeric receptors than at homomeric GluR1o, suggesting that its efficacy and/or desensitisation properties are different at GluR1o vs. GluR3o.

Comparison of the agonist binding site of homomeric, heteromeric, and chimeric GluR1(o) and GluR3(o) AMPA receptors.

A series of AMPA [(R,S)-alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid] analogues were evaluated for activity at homomeric, heteromeric, and chimeric rat GluR1(o) and GluR3(o) receptors expressed in Xenopus oocytes, using the two-electrode voltage clamp technique. The formation of heteromeric receptor complexes was demonstrated by cross-immunoprecipitation of both subunits from solubilized oocyte membranes. The AMPA analogue ACPA [(R,S)-2-amino-3(3-carboxy-5-methyl-4-isoxazolyl)propionic acid] was the most potent and selective agonist tested at GluR1(o) and GluR3(o), with a 10-fold selectivity for GluR3(o). ACPA showed an intermediate potency at both the GluR1(o) + 3(o) heteromeric complex as well as at the homomeric chimeric receptors. These experiments suggest that for receptor activation, agonist binding occurs between the interface of the GluR1 and GluR3 subunits in the heteromeric channel complex, perhaps between the S1 region of one subunit and the S2 region of another. Also, it seems that 1) electronegative group substitutions on the isoxazole ring of AMPA and 2) decreasing the pKa of the sub stituent at position 3 play a major role in determining the degree of receptor activation under steady-state conditions. Future studies will examine the effects of single amino acid mutations in these receptors, giving a more precise localization of the agonist binding site.