Group I metabotropic glutamate receptors activate a calcium-sensitive transient receptor potential-like conductance in rat hippocampus

Abstract

In CA3 pyramidal neurons from organotypic slice cultures, activation of G(q)-coupled group I metabotropic glutamate receptors (mGluRs) induces a non-selective cationic conductance that enhances excitability. We have found that this response shares several properties with conductances that are mediated by the transient receptor potential (TRP) family of ion channels, including inhibition by La(3+), 2-aminoethoxydiphenylborane (2APB), cis-N-(2-phenylcyclopentyl)azacyclotridec-1-en-2-amine (MDL 12,330A) and a doubly rectifying current-voltage relationship. Stimulation of mGluR1 and mGluR5 converged to activate the TRP-like conductance in a synergistic manner, and activation of either subtype alone produced only a fraction of the normal response. Activation of the cationic current required elevated intracellular Ca(2+). Chelating intracellular Ca(2+) or blocking Ca(2+) entry through voltage-gated Ca(2+) channels attenuated responses to the activation of mGluRs. Conversely, raising intracellular Ca(2+) potentiated mGluR activation of the TRP-like conductance. Under control conditions, blocking G protein activation using intracellular GDPbetaS with or without N-(2, 6-dimethylphenylcarbamoylmethyl) triethylammonium chloride (QX-314) prevented mGluR-mediated activation of the TRP-like conductance. Following G protein blockade, however, the coupling between mGluRs 1 and/or 5 and the TRP-like conductance was rescued by increasing intracellular Ca(2+). This suggests that a G protein-independent signalling pathway is also activated by group I mGluRs. Such a pathway may represent an alternative transduction mechanism to maintain metabotropic responses under conditions where G proteins are functionally uncoupled from their cognate receptors.

Figure 1. Transient receptor potential (TRP)-like channels conduct the current induced by (S)-3, 5-dihydroxyphenylglycine (DHPG) in CA3 pyramidal cells

A, sample response to a 30 s application of 10 µm DHPG in a cell voltage clamped at −50 mV. B, the average subtracted I-V relationship of the response to DHPG, calculated from 3 s voltage ramps (+40 to −120 mV, n = 5), indicates that the response is associated with an increase in a mixed cationic conductance. Inset is the peak current in response to DHPG vs. different holding potentials in a typical cell. Superimposed on the I-V relationship is the change in Ca²⁺ levels (ΔF/F) with increasing holding potential from cells loaded with Oregon Green BAPTA-2 (circles, n = 4). Intracellular Ca²⁺ rose with increasing voltage beginning from −60 mV and began to plateau at −40 mV, close to the peak of the inward current. C, 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB), an inhibitor of Cl⁻ channels and anion exchangers that also inhibits some cationic conductances, inhibited the DHPG-induced current. D, exchanging intracellularCH³SO³⁻ for Cl⁻ to reverse the direction of current flow through Cl⁻ channels and anion exchangers had no effect on the response to DHPG. E, La³⁺, an antagonist of both TRP channels and Ca²⁺ channels, inhibited responses to DHPG. F, the TRP channel and adenylyl cyclase antagonist cis-N-(2-phenylcyclopentyl)azacyclotridec-1-en-2-amine (MDL 12,330A) inhibited the response to DHPG. G, 2-aminoethoxydiphenylborane (2APB), which inhibits TRP channels and inositol trisphosphate receptor (IP₃R)- mediated events, inhibited the DHPG response. In this and subsequent figures, traces of control data (cntl) and traces obtained after drug application are from the same cell voltage clamped at −50 mV. The line below traces indicates time of DHPG entering the bath. The n values are given on the figures; n =x indicates that the same x cells are included in each condition. Numbers in parentheses indicate either that a subpopulation is included, especially after washes, or that different cells were used in each condition. Pooled data are shown as means ±s.e.m. *P≤ 0.05.

Figure 2. Metabotropic glutamate receptors (mGluRs) 1 (mGluR1) and 5 (mGluR5) contribute synergistically to activate a cationic conductance in CA3 pyramidal cells

A, top traces show responses to DHPG in control conditions and bottom traces show responses to DHPG after application of either the mGluR1 antagonist LY367385 (LY), the mGluR5 antagonist 2-methyl-6-(phenylethynyl)pyridine hydrochloride (MPEP), or both. B, pooled data show that blocking either mGluR1 or mGluR5 reduced the response to DHPG by more than 50 % in CA3 pyramidal neurons. Only the block by the competitive antagonist LY367385 was reversible.

Figure 3. Intracellular GDPβS (1 mm) prevents activation of the cationic current to DHPG

A₁, with intracellular Cs⁺ exchanged for K⁺, the GABA_B receptor agonist baclofen, applied within 2 min after obtaining whole-cell access, induced a G protein-dependent K⁺ current. This response was completely blocked after 12–60 min dialysis with GDPβS. A₂, subsequent application of DHPG induced no response in CA3 pyramidal cells. B, pooled responses to DHPG from cells with intracellular K⁺ and either GTP or GDPβS.

Figure 4. The response to DHPG is Ca²⁺ dependent

A, including 40 mm BAPTA in the recording pipette suppressed the inward current induced by DHPG. B, blocking voltage-gated Ca²⁺ channels (VGCCs) with Cd²⁺ strongly reduced the inward current. C, substituting Ba²⁺ for extracellular Ca²⁺ prevented activation of the inward current by DHPG. D, increasing extracellular K⁺ from 2.7 mm (2.7K)to 8 mm (8K) increased the response to DHPG. E, in the continuous presence of 100 µm Cd²⁺+ 50 µm Ni²⁺, increasing the extracellular K⁺ concentration no longer enhanced responses to DHPG. F, the increase in inward current by raising extracellular K⁺ was also prevented by a cocktail of toxins that block VGCCs (see text).

Figure 5. Raising intracellular Ca²⁺ rescues the response to DHPG following G protein blockade

A, sample traces from one cell recorded with intracellular K⁺ and 1 mm GDPβS are shown in chronological order. A_{1, 2}, the loss of response to baclofen indicates G protein blockade. A₃, following G protein blockade, responses to DHPG were blocked. A₄, raising intracellular Ca²⁺ by increasing extracellular K⁺ rescued the response to selective activation of mGluR5 (DHPG + LY367385). A₅, rescue is also observed by activation of both group I mGluRs after washing LY367385 from the slice. A₆, response in 8 mm K⁺ to selective activation of mGluR1 (DHPG + MPEP). B₁, pooled data with GTP, GDPβS or GDPβS plus N-(2,6-dimethylphenylcarbamoylmethyl) triethylammonium chloride (QX-314; 5 mm) showing the block of responses to DHPG in 2.7 mm K⁺, potentiation of the response in GTP cells with 8 mm K⁺ and rescue of the response after G protein blockade. B₂, with intracellular Cs⁺, GDPβS still blocked the response to DHPG in 2.7 mm K⁺ and responses were rescued with 8 mm K⁺. C, with intracellular GDPβS, responses to baclofen recorded within 3 min of gaining whole-cell access were abolished within 12–60 min. Raising extracellular K⁺ to 8 mm did not affect G protein blockade by GDPβS. With QX-314 added to the GDPβS-containing solution, baclofen responses were blocked within 10–36 min, and again were not affected by the rise in intracellular Ca²⁺ induced by 8 mm K⁺. D, pooled data from five cells in which responses to DHPG with the mGluR1- and mGluR5-selective antagonists were obtained in 8 mm K⁺ following G protein blockade. E, rescue of responses to DHPG by 8 mm K⁺ was not affected by the phospholipase C antagonist U73122 (10 µm). In two cells, G proteins were blocked by GDPβS, and in five cells they were blocked by GDPβS plus QX-314. F, following G protein blockade, the L-type Ca²⁺ channel agonist BAY K8644 also rescued the response to DHPG. Traces are from one cell, with baclofen responses abolished by GDPβS. G, BAY K8644 increased intracellular Ca²⁺ levels (holding potential −50 mV). A typical CA3 pyramidal cell showing the region of interest along with the simultaneous Ca²⁺ and electrophysiological recordings.