Journal of Neuroscience, Vol 11, 3610-3623, Copyright © 1991 by Society for Neuroscience
Denervation does not alter the number of neuronal bungarotoxin binding sites on autonomic neurons in the frog cardiac ganglion
PB Sargent, GK Bryan, LC Streichert and EN Garrett
Department of Stomatology (Oral Biology), University of California, San Francisco 94143-0512.
The binding of neuronal bungarotoxin (n-BuTX; also known as bungarotoxin
3.1, kappa-bungarotoxin, and toxin F) was analyzed in normal and denervated
parasympathetic cardiac ganglia of the frog Rana pipiens, n-BuTX blocks
both EPSPs and ACh potentials at 5-20 nM, as determined by intracellular
recording techniques. Scatchard analysis on homogenates indicates that
cardiac ganglia have two classes of binding sites for 125I-n-BuTX: a
high-affinity site with an apparent dissociation constant (Kd,app) of 1.7
nM and a Bmax (number of binding sites) of 3.8 fmol/ganglion and a
low-affinity site with a Kd,app of 12 microM and a Bmax of 14
pmol/ganglion. alpha-Bungarotoxin does not appear to interfere with the
binding of 125I-n-BuTX to either site. The high-affinity binding site is
likely to be the functional nicotinic ACh receptor (AChR), given the
similarity between its affinity for 125I-n- BuTX and the concentration of
n-BuTX required to block AChR function. Light microscopic autoradiographic
analysis of 125I-n-BuTX binding to the ganglion cell surface reveals that
toxin binding is concentrated at synaptic sites, which were identified
using a synaptic vesicle-specific antibody. Scatchard analysis of
autoradiographic data reveals that 125I- n-BuTX binding to the neuronal
surface is saturable and has a Kd,app similar to that of the high-affinity
binding site characterized in homogenates. Surface binding of 125I-n-BuTX
is blocked by nicotine, carbachol, and d-tubocurarine (IC50 less than 20
microM), but not by atropine (IC50 greater than 10 mM). Denervation of the
heart increases the ACh sensitivity of cardiac ganglion cells but has no
effect upon the number of high-affinity binding sites for 125I-n-BuTX in
tissue homogenates. Moreover, autoradiographic analysis indicates that
denervation does not alter the number of 125I-n-BuTX binding sites on the
ganglion cell surface. n-BuTX is as effective in reducing ganglion cell
responses to ACh in denervated ganglia as it is in normally innervated
ganglia. These results suggest that denervation alters neither the total
number of nicotinic AChRs in the cardiac ganglion nor the number found on
the surface of ganglion cells. These autonomic neurons thus respond
differently to denervation than do skeletal myofibers. The increase in ACh
sensitivity displayed by cardiac ganglion cells upon denervation cannot be
explained by changes in AChR number.