Diabetes Depresses Synaptic Transmission in Sympathetic Ganglia by Inactivating nAChRs through a Conserved Intracellular Cysteine Residue

Abstract

Most people with diabetes develop severe complications of the autonomic nervous system; yet, the underlying causes of many diabetic-induced dysautonomias are poorly understood. Here we explore the idea that these dysautonomias results, in part, from a defect in synaptic transmission. To test this idea, we investigated cultured sympathetic neurons and show that hyperglycemia inactivates nAChRs through a mechanism involving an elevation in reactive oxygen species and an interaction with highly conserved cysteine residues located near the intracellular mouth of the nAChR channel. Consistent with this, we show that diabetic mice have depressed ganglionic transmission and reduced sympathetic reflexes, whereas diabetic mice expressing mutant postsynaptic nAChRs that lack the conserved cysteine residues on the alpha3 subunit have normal synaptic transmission in sympathetic ganglia and normal sympathetic reflexes. Our work suggests a new model for diabetic-induced dysautonomias and identifies ganglionic nAChRs as targets of hyperglycemia-induced downstream signals.

Synapses on Sympathetic Neurons and Parasympathetic Neurons Differ in Their Vulnerability to Diabetes

“…For adrenal medullae, we used Ca 2ϩ -and Mg 2ϩ -free HBSS. The resulting cell suspension was washed twice in serum-containing medium and plated on laminin-coated coverslips attached to modified 35 mm culture dishes (Campanucci et al, 2008). Sympathetic neurons were grown in media consisting of L-15 supplemented with 5 mM glucose, vitamins, cofactors, penicillin-streptomycin, 5% rat serum, and 25 ng/ml nerve growth factor (NGF).…”

“…ACh-evoked currents were measured with whole-cell patch clamp as described previously (Campanucci et al, 2008). Membrane currents were recorded with a VE-2 amplifier (Alembic Instruments) at room temperature and were filtered and analyzed offline with Igor software.…”

A Highly Conserved Cytoplasmic Cysteine Residue in the α4 Nicotinic Acetylcholine Receptor Is Palmitoylated and Regulates Protein Expression

“…It is interesting to note that the cysteine that is palmitoylated in our study has been suggested to undergo reactive oxygen species-regulated use-dependent inactivation in the ␣3␤4 nAChR (14). The authors propose that use-dependent inactivation of the ␣3␤4 nAChR occurs after the receptors have been repeatedly exposed to agonist over time due to mild elevations in reactive oxygen species (14,21,22).…”

“…The authors propose that use-dependent inactivation of the ␣3␤4 nAChR occurs after the receptors have been repeatedly exposed to agonist over time due to mild elevations in reactive oxygen species (14,21,22). Receptor use can also enhance the depalmitoylation of proteins (23)(24)(25)(26)(27).…”

“…The cysteine in the M1-M2 loop is palmitoylated and is conserved in the ␣2, ␣3, ␣4, ␣6, ␤2, and ␤4 nAChR subunits. The homologous cysteine previously has been associated with use-dependent inactivation of ␣3-containing nAChRs in hyperglycemic and oxidative conditions (14). When this cysteine is mutated to a serine, creating a depalmitoylated mutant, total protein expression is decreased, whereas cell surface expression increases.…”