Amy B. Harkins

Neuronal alpha-bungarotoxin receptors differ structurally from other nicotinic acetylcholine receptors

We have characterized the α-bungarotoxin receptors (BgtRs) found on the cell surface of undifferentiated pheochromocytoma (PC12) cells. The PC12 cells express a homogeneous population of α7-containing receptors that bind α-Bgt with high affinity (Kd = 94 pm). The BgtRs mediate most of the response elicited by nicotine, because the BgtR-specific antagonists methyllycaconitine and α-Bgt block ∼90% of the whole-cell current. The binding of nicotinic agonists to cell-surface BgtRs was highly cooperative with four different agonists showing Hill coefficients in the range of 2.3–2.4. A similar agonist binding cooperativity was observed for BgtR homomers formed from chimeric α7/5HT3 subunits expressed in tsA 201 cells. Two classes of agonist binding sites, in the ratio of 4:1 for PC12 cell BgtRs and 3:1 for α7/5HT3 BgtRs, were revealed by bromoacetylcholine alkylation of the reduced sites on both PC12 BgtRs and α7/5HT3 BgtRs. We conclude from this data that PC12 BgtRs and α7/5HT3 homomers contain at least three distinguishable agonist binding sites and thus are different from other nicotinic receptors.

Cell death in weaver mouse cerebellum.

Mice with theweaver mutation exhibit an uneven weave to their gait, ataxia, mild locomotor hyperactivity and, occasionally, tonic-clonic seizures. A single amino acid mutation in a G-protein coupled, inwardly rectifying K+ channel, GIRK2, gives rise to the symptoms seen in theweaver mice. Two areas of the brain are primarily affected. Cerebellar granule cell neurons die soon after birth and dopaminergic neurons are severely depleted in the substantia nigra. In this article we review recent studies of wild-type and mutant GIRK channels found in native cells or introduced into expression systems. We also review two models that explain some of the details leading to the neuronal cell death observed inweaver mice.

Activation of purinergic receptors by ATP inhibits secretion in bovine adrenal chromaffin cells

Autoinhibition is a common mechanism observed in neurons to regulate neurotransmission. Released neurotransmitter interacts with presynaptic autoreceptors to inhibit subsequent release. The requisite elements for autoinhibition are present in chromaffin cells: secretory granules contain millimolar levels of ATP which is coreleased with catecholamines upon stimulation and the cells express purinergic receptors. We were interested to determine whether autoinhibition produced by ATP binding to purinergic receptors plays an important role in catecholamine release from chromaffin cells. In these studies, short depolarizations were used to elicit transmitter release measured by membrane capacitance. We find that stimulation of chromaffin cells results in the release of endogenous ATP which may suppress Ca(2+) channel currents and secretion. In the presence of a maximal concentration of ATP, both the amount of secretion and the maximal rate of release are about half that observed in the absence of ATP. ATP-mediated inhibition of secretion was blocked by Reactive Blue-2 suggesting the involvement of P(2Y) purinergic receptors. Prepulses to positive potentials that relieve the Ca(2+) channel block largely relieve the inhibition of secretion. Furthermore, when secretion is plotted as a function of Ca(2+) influx there is no apparent change in the relationship between control cells and those stimulated in the presence of ATP and prepulses. These results suggest that ATP diminishes secretion by inhibiting Ca(2+) influx into the cells. Our results indicate that feedback inhibition by ATP, mediated primarily by Ca(2+) channels, may be an important regulator of catecholamine release in chromaffin cells.

Light Activation of Calcein Inhibits Vesicle Release of Catecholamines

Calcein, a fluorescent fluid phase marker, has been used to track and visualize cellular processes such as synaptic vesicle fusion. It is also the fluorophore for live cells in the commonly used Live/Dead viability assay. In pilot studies designed to determine fusion pore open size and vesicle movement in secretory cells, imaging analysis revealed that calcein reduced the number of vesicles released from the cells when stimulated with nicotine. Using amperometry to detect individual vesicle release events, we show that when calcein is present in the media, the number of vesicles that fuse with the cellular membrane is reduced when cells are stimulated with either nicotine or high K+. Experimentally, amperometric electrodes are not undergoing fouling in the presence of calcein. We hypothesized that calcein, when activated by light, releases reactive oxygen species that cause a reduction in secreted vesicles. We show that when calcein is protected from light during experimentation, little to no reduction of vesicle secretion occurred. Therefore, photoactivated calcein can cause deleterious results for measurements of cellular processes, likely to be the result of release of reactive oxygen species.

Titration of Synaptotagmin I Expression Differentially Regulates Release of Neuropeptide Y and Norepinephrine

Synaptotagmin (syt) I is a primary regulator of Ca2+-dependent vesicle secretion. Pheochromocytoma (PC12) cells are used as an immortalized cell model system for neurons to study regulated vesicle release. In this study, we stably transfected multiple PC12 cell lines with a single plasmid that contains a syt I-targeting short hairpin RNA to knockdown expression of syt I. Stable cells were selected, expanded, and tested for stable incorporation of the plasmid with PCR and for specific targeting of syt I with immunoblot analysis. As previously reported (Cahill et al., 2007), individual stable cell lines derived from incorporation of a single short hairpin RNA expressed varying levels of syt I knockdown. This variability offers the advantages of studying the functional effects of graded levels of syt I protein expression in regulated release of transmitter. We measured Ca2+-stimulated release of two transmitters, neuropeptide Y (NPY) and norepinephrine (NE). NPY was measured by an enzyme-immunoassay after depolarizing with 50 mM K+ solution. Stable cell lines that expressed 50-60% of control levels of syt I exhibited NPY release that were similar to control cells. NPY release was reduced to about 18% of control cell release when expression of syt I was reduced to ∼20%, and NPY release was abolished when syt I expression was abolished. Stimulated NE release from single vesicles was measured by carbon-fiber amperometry. Unlike NPY release, NE release ranged from 50-100% compared to control release, but was not abolished even for the cells that did not express syt I. These results show that syt I is required for NPY release, but NE release is only partially dependent on syt I expression.