Mutations in M2 alter the selectivity of the mouse nicotinic acetylcholine receptor for organic and alkali metal cations

We measured the permeability ratios (PX/PNa) of 3 wild-type, 1 hybrid, 2 subunit-deficient, and 22 mutant nicotinic receptors expressed in Xenopus oocytes for alkali metal and organic cations using shifts in the bi-ionic reversal potential of the macroscopic current. Mutations at three positions (2', 6', 10') in M2 affected ion selectivity. Mutations at position 2' (alpha Thr244, beta Gly255, gamma Thr253, delta Ser258) near the intracellular end of M2 changed the organic cation permeability ratios as much as twofold and reduced PCs/PNa and PK/PNa by 16-18%. Mutations at positions 6' and 10' increased the glycine ethyl ester/Na+ and glycine methyl ester/Na+ permeability ratios. Two subunit alterations also affected selectivity: omission of the delta subunit reduced PCs/PNa by 16%, and substitution of Xenopus delta for mouse delta increased Pguanidinium/PNa more than twofold and reduced PCs/PNa by 34% and PLi/PNa by 20%. The wild-type mouse receptor displayed a surprising interaction with the primary ammonium cations; relative permeability peaked at a chain length equal to four carbons. Analysis of the organic permeability ratios for the wild-type mouse receptor shows that (a) the diameter of the narrowest part of the pore is 8.4 A; (b) the mouse receptor departs significantly from size selectivity for monovalent organic cations; and (c) lowering the temperature reduces Pguanidinium/PNa by 38% and Pbutylammonium/PNa more than twofold. The results reinforce present views that positions -1' and 2' are the narrowest part of the pore and suggest that positions 6' and 10' align some permeant organic cations in the pore in an interaction similar to that with channel blocker, QX-222

Tris+/Na+ permeability ratios of nicotinic acetylcholine receptors are reduced by mutations near the intracellular end of the M2 region

Tris+/Na+ permeability ratios were measured from shifts in the biionic reversal potentials of the macroscopic ACh-induced currents for 3 wild- type (WT), 1 hybrid, 2 subunit-deficient, and 25 mutant nicotinic receptors expressed in Xenopus oocytes. At two positions near the putative intracellular end of M2, 2' (alpha Thr244, beta Gly255, gamma Thr253, delta Ser258) and -1', point mutations reduced the relative Tris+ permeability of the mouse receptor as much as threefold. Comparable mutations at several other positions had no effects on relative Tris+ permeability. Mutations in delta had a greater effect on relative Tris+ permeability than did comparable mutations in gamma; omission of the mouse delta subunit (delta 0 receptor) or replacement of mouse delta with Xenopus delta dramatically reduced relative Tris+ permeability. The WT mouse muscle receptor (alpha beta gamma delta) had a higher relative permeability to Tris+ than the wild-type Torpedo receptor. Analysis of the data show that (a) changes in the Tris+/Na+ permeability ratio produced by mutations correlate better with the hydrophobicity of the amino acid residues in M2 than with their volume; and (b) the mole-fraction dependence of the reversal potential in mixed Na+/Tris+ solutions is approximately consistent with the Goldman- Hodgkin-Katz voltage equation. The results suggest that the main ion selectivity filter for large monovalent cations in the ACh receptor channel is the region delimited by positions -1' and 2' near the intracellular end of the M2 helix

A G protein-gated K channel is activated via beta 2-adrenergic receptors and G beta gamma subunits in Xenopus oocytes

In many tissues, inwardly rectifying K channels are coupled to seven- helix receptors via the Gi/Go family of heterotrimeric G proteins. This activation proceeds at least partially via G beta gamma subunits. These experiments test the hypothesis that G beta gamma subunits activate the channel even if released from other classes of heterotrimeric G proteins. The G protein-gated K channel from rat atrium, KGA/GIRK1, was expressed in Xenopus oocytes with various receptors and G proteins. The beta 2-adrenergic receptor (beta 2AR), a Gs-linked receptor, activated large KGA currents when the alpha subunit, G alpha s, was also overexpressed. Although G alpha s augmented the coupling between beta 2AR and KGA, G alpha s also inhibited the basal, agonist-independent activity of KGA. KGA currents stimulated via beta 2AR activated, deactivated, and desensitized more slowly than currents stimulated via Gi/Go-linked receptors. There was partial occlusion between currents stimulated via beta 2AR and the m2 muscarinic receptor (a Gi/Go-linked receptor), indicating some convergence in the mechanism of activation by these two receptors. Although stimulation of beta 2AR also activates adenylyl cyclase and protein kinase A, activation of KGA via beta 2AR is not mediated by this second messenger pathway, because direct elevation of intracellular cAMP levels had no effect on KGA currents. Experiments with other coexpressed G protein alpha and beta gamma subunits showed that (a) a constitutively active G alpha s mutant did not suppress basal KGA currents and was only partially as effective as wild type G alpha s in coupling beta 2AR to KGA, and (b) beta gamma subunits increased basal KGA currents. These results reinforce present concepts that beta gamma subunits activate KGA, and also suggest that beta gamma subunits may provide a link between KGA and receptors not previously known to couple to inward rectifiers

Allosteric Control of Gating and Kinetics at P2X₄ Receptor Channels

The CNS abundantly expresses P2X receptor channels for ATP; of these the most widespread in the brain is the P2X₄ channel. We show that ivermectin (IVM) is a specific positive allosteric effector of heterologously expressed P2X₄ and possibly of heteromeric P2X₄/P2X₆channels, but not of P2X₂, P2X₃, P2X₂/P2X₃, or P2X₇ channels. In the submicromolar range (EC₅₀, ∼250 nM) the action of IVM was rapid and reversible, resulting in increased amplitude and slowed deactivation of P2X₄ channel currents evoked by ATP. IVM also markedly increased the potency of ATP and that of the normally low-potency agonist α,β-methylene-ATP in a use- and voltage-independent manner without changing the ion selectivity of P2X₄ channels. Therefore, IVM evokes a potent pharmacological gain-of-function phenotype that is specific for P2X₄ channels. We also tested whether IVM could modulate endogenously expressed P2X channels in the adult trigeminal mesencephalic nucleus and hippocampal CA1 neurons. Surprisingly, IVM produced no significant effect on the fast ATP-evoked inward currents in either type of neuron, despite the fact that IVM modulated P2X₄ channels heterologously expressed in embryonic hippocampal neurons. These results suggest that homomeric P2X₄ channels are not the primary subtype of P2X receptor in the adult trigeminal mesencephalic nucleus and in hippocampal CA1 neurons

Allosteric Control of Gating and Kinetics at P2X₄ Receptor Channels

The CNS abundantly expresses P2X receptor channels for ATP; of these the most widespread in the brain is the P2X₄ channel. We show that ivermectin (IVM) is a specific positive allosteric effector of heterologously expressed P2X₄ and possibly of heteromeric P2X₄/P2X₆channels, but not of P2X₂, P2X₃, P2X₂/P2X₃, or P2X₇ channels. In the submicromolar range (EC₅₀, ∼250 nM) the action of IVM was rapid and reversible, resulting in increased amplitude and slowed deactivation of P2X₄ channel currents evoked by ATP. IVM also markedly increased the potency of ATP and that of the normally low-potency agonist α,β-methylene-ATP in a use- and voltage-independent manner without changing the ion selectivity of P2X₄ channels. Therefore, IVM evokes a potent pharmacological gain-of-function phenotype that is specific for P2X₄ channels. We also tested whether IVM could modulate endogenously expressed P2X channels in the adult trigeminal mesencephalic nucleus and hippocampal CA1 neurons. Surprisingly, IVM produced no significant effect on the fast ATP-evoked inward currents in either type of neuron, despite the fact that IVM modulated P2X₄ channels heterologously expressed in embryonic hippocampal neurons. These results suggest that homomeric P2X₄ channels are not the primary subtype of P2X receptor in the adult trigeminal mesencephalic nucleus and in hippocampal CA1 neurons

Assembly of α4β2 Nicotinic Acetylcholine Receptors Assessed with Functional Fluorescently Labeled Subunits: Effects of Localization, Trafficking, and Nicotine-Induced Upregulation in Clonal Mammalian Cells and in Cultured Midbrain Neurons

Fura-2 recording of Ca^(2+) influx was used to show that incubation in 1 μM nicotine (2-6 d) upregulates several pharmacological components of acetylcholine (ACh) responses in ventral midbrain cultures, including a MLA-resistant, DHβE-sensitive component that presumably corresponds to α4β2 receptors. To study changes in α4β2 receptor levels and assembly during this upregulation, we incorporated yellow and cyan fluorescent proteins (YFPs and CFPs) into the α4 or β2 M3-M4 intracellular loops, and these subunits were coexpressed in human embryonic kidney (HEK) 293T cells and cultured ventral midbrain neurons. The fluorescent receptors resembled wild-type receptors in maximal responses to ACh, dose-response relations, ACh-induced Ca^(2+) influx, and somatic and dendritic distribution. Transfected midbrain neurons that were exposed to nicotine (1 d) displayed greater levels of fluorescent α4 and β2 nicotinic ACh receptor (nAChR) subunits. As expected from the hetero-multimeric nature of α4β2 receptors, coexpression of the α4-YFP and β2-CFP subunits resulted in robust fluorescence resonance energy transfer (FRET), with a FRET efficiency of 22%. In midbrain neurons, dendritic α4β2 nAChRs displayed greater FRET than receptors inside the soma, and in HEK293T cells, a similar increase was noted for receptors that were translocated to the surface during PKC stimulation. When cultured transfected midbrain neurons were incubated in 1 μMnicotine, there was increased FRET in the cell body, denoting increased assembly of α4β2 receptors. Thus, changes in α4β2 receptor assembly play a role in the regulation of α4β2 levels and responses in both clonal cell lines and midbrain neurons, and the regulation may result from Ca^(2+)-stimulated pathways

Cell-specific posttranslational events affect functional expression at the plasma membrane but not tetrodotoxin sensitivity of the rat brain IIA sodium channel α-subunit expressed in mammalian cells

The rat brain IIA Na⁺ channel alpha-subunit was expressed and studied in mammalian cells. Cells were infected with a recombinant vaccinia virus (VV) carrying the bacteriophage T7 RNA polymerase gene and were transfected with cDNA encoding the IIA Na⁺ channel α-subunit under control of a T7 promoter. Whole-cell patch-clamp recording showed that functional IIA channels were expressed efficiently (~10 channels/ µm² in approximately 60% of cells) in Chinese hamster ovary (CHO) cells and in neonatal rat ventricular myocytes but were expressed poorly in undifferentiated BC₃H1 cells and failed to express in Ltk⁻ cells. However, voltage-dependent Drosophila Shaker H4 K⁺ channels and Escherichia coli β-galactosidase were expressed efficiently in all four cell types with VV vectors. Because RNA synthesis probably occurs without major differences in the cytoplasm of all infected cell types under the control of the T7 promoter and T7 polymerase, we conclude that cell type-specific expression of the Na⁺ channel probably reflects differences at posttranslational steps. The gating properties of the IIA Na⁺ currents expressed in cardiac myocytes differed from those expressed in CHO cells; most noticeably, the IIA Na⁺ currents displayed more rapid macroscopic inactivation when expressed in cardiac myocytes. These differences also suggest cell- specific posttranslational modifications. IIA channels were blocked by ~90% by 90 nM TTX when expressed either in CHO cells or in cardiac myocytes; the latter also continued to display endogenous TTX- resistant Na⁺ currents. Therefore, the TTX binding site of the channel is not affected by cell-specific modifications and is encoded by the primary amino acid sequence

Increased Sensitivity to Agonist-Induced Seizures, Straub Tail, and Hippocampal Theta Rhythm in Knock-In Mice Carrying Hypersensitive α4 Nicotinic Receptors

We studied a strain of exon replacement mice (“L9′S knock-in”) whose α4 nicotinic receptor subunits have a leucine to serine mutation in the M2 region, 9′ position (Labarca et al., 2001); this mutation renders α4-containing receptors hypersensitive to agonists. Nicotine induced seizures at concentrations (1 mg/kg) approximately eight times lower in L9′S than in wild-type (WT) littermates. At these concentrations, L9′S but not WT showed increases in EEG amplitude and theta rhythm. L9′S mice also showed higher seizure sensitivity to the nicotinic agonist epibatidine, but not to the GABA_Areceptor blocker and proconvulsant bicuculline. Dorsiflexion of the tail (Straub tail) was the most sensitive nicotine effect found in L9′S mice (0.1 mg/kg). The L9′S mice were hypersensitive to galanthamine- and tacrine-induced seizures and Straub tails. There were no apparent neuroanatomical differences between L9′S and WT mice in several brain regions. [125I]Epibatidine binding to brain membranes showed that the mutant allele was expressed at ∼25% of WT levels, presumably because of the presence of a neomycin selection cassette in a nearby intron. ^(86)Rb efflux experiments on brain synaptosomes showed an increased fraction of function at low agonist concentrations in L9′S mice. These data support the possible involvement of gain-of-function α4 receptors in autosomal dominant nocturnal frontal-lobe epilepsy