Neuronal α-Bungarotoxin Receptors Differ Structurally from Other Nicotinic Acetylcholine Receptors

Abstract

We have characterized the ␣-bungarotoxin receptors (BgtRs) found on the cell surface of undifferentiated pheochromocytoma (PC12) cells. The PC12 cells express a homogeneous population of ␣7-containing receptors that bind ␣-Bgt with high affinity (K d ϭ 94 pM). The BgtRs mediate most of the response elicited by nicotine, because the BgtR-specific antagonists methyllycaconitine and ␣-Bgt block ϳ90% 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 ␣7/5HT3 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 ␣7/5HT3 BgtRs, were revealed by bromoacetylcholine alkylation of the reduced sites on both PC12 BgtRs and ␣7/5HT3 BgtRs. We conclude from this data that PC12 BgtRs and ␣7/5HT3 homomers contain at least three distinguishable agonist binding sites and thus are different from other nicotinic receptors.

Two distinct classes of functional α7‐containing nicotinic receptor on rat superior cervical ganglion neurons

“…Moreover, a partial reversal of the MLA block could be demonstrated with continued wash (Fig. 6) as previously seen for MLA blockade of α7‐nAChR responses in rat PC12 cells (Rangwala et al 1997). It was not possible to use intracellular dialysis to test the effects of anti‐α7 mAbs on the rapidly decaying response because it underwent significant rundown with time of recording when conventional patch clamp methods were used to dialyse the interior of the cells.…”

Two distinct classes of functional α7‐containing nicotinic receptor on rat superior cervical ganglion neurons

“…Moreover, a partial reversal of the MLA block could be demonstrated with continued wash (Fig. 6) as previously seen for MLA blockade of α7‐nAChR responses in rat PC12 cells (Rangwala et al 1997). It was not possible to use intracellular dialysis to test the effects of anti‐α7 mAbs on the rapidly decaying response because it underwent significant rundown with time of recording when conventional patch clamp methods were used to dialyse the interior of the cells.…”

α-Bungarotoxin Receptors Contain α7 Subunits in Two Different Disulfide-Bonded Conformations

“…4-6). These data are consistent with ␣7 subunits complexing into pentamers, similar to ␣7/5HT 3 subunit receptors (Rangwala et al, 1997) and other characterized AChRs (Karlin and Akabas, 1995). For ␣7 subunits, a large number of the multimers was present even on reducing gels or alkylated with NEM, though alkylation did disperse some of the larger multimers ( Fig.…”

alpha-Bungarotoxin Receptors Contain alpha7 Subunits in Two Different Disulfide-bonded Conformations

“…Bands corresponding to subunit dimers, trimers, tetramers, and pentamers were observed, but there were no complexes larger than pentamers (see . These data are consistent with ␣7 subunits complexing into pentamers, similar to ␣7/5HT 3 subunit receptors (Rangwala et al, 1997) and other characterized AChRs (Karlin and Akabas, 1995). For ␣7 subunits, a large number of the multimers was present even on reducing gels or alkylated with NEM, though alkylation did disperse some of the larger multimers ( Fig.…”

Two distinct classes of functional α7‐containing nicotinic receptor on rat superior cervical ganglion neurons

“…Moreover, a partial reversal of the MLA block could be demonstrated with continued wash (Fig. 6) as previously seen for MLA blockade of α7‐nAChR responses in rat PC12 cells (Rangwala et al 1997). It was not possible to use intracellular dialysis to test the effects of anti‐α7 mAbs on the rapidly decaying response because it underwent significant rundown with time of recording when conventional patch clamp methods were used to dialyse the interior of the cells.…”

α-Bungarotoxin Receptors Contain α7 Subunits in Two Different Disulfide-Bonded Conformations

“…4-6). These data are consistent with ␣7 subunits complexing into pentamers, similar to ␣7/5HT 3 subunit receptors (Rangwala et al, 1997) and other characterized AChRs (Karlin and Akabas, 1995). For ␣7 subunits, a large number of the multimers was present even on reducing gels or alkylated with NEM, though alkylation did disperse some of the larger multimers ( Fig.…”

alpha-Bungarotoxin Receptors Contain alpha7 Subunits in Two Different Disulfide-bonded Conformations

“…Bands corresponding to subunit dimers, trimers, tetramers, and pentamers were observed, but there were no complexes larger than pentamers (see . These data are consistent with ␣7 subunits complexing into pentamers, similar to ␣7/5HT 3 subunit receptors (Rangwala et al, 1997) and other characterized AChRs (Karlin and Akabas, 1995). For ␣7 subunits, a large number of the multimers was present even on reducing gels or alkylated with NEM, though alkylation did disperse some of the larger multimers ( Fig.…”

Two distinct classes of functional α7‐containing nicotinic receptor on rat superior cervical ganglion neurons

“…Moreover, a partial reversal of the MLA block could be demonstrated with continued wash (Fig. 6) as previously seen for MLA blockade of α7‐nAChR responses in rat PC12 cells (Rangwala et al 1997). It was not possible to use intracellular dialysis to test the effects of anti‐α7 mAbs on the rapidly decaying response because it underwent significant rundown with time of recording when conventional patch clamp methods were used to dialyse the interior of the cells.…”

α-Bungarotoxin Receptors Contain α7 Subunits in Two Different Disulfide-Bonded Conformations

“…4-6). These data are consistent with ␣7 subunits complexing into pentamers, similar to ␣7/5HT 3 subunit receptors (Rangwala et al, 1997) and other characterized AChRs (Karlin and Akabas, 1995). For ␣7 subunits, a large number of the multimers was present even on reducing gels or alkylated with NEM, though alkylation did disperse some of the larger multimers ( Fig.…”

alpha-Bungarotoxin Receptors Contain alpha7 Subunits in Two Different Disulfide-bonded Conformations

“…Bands corresponding to subunit dimers, trimers, tetramers, and pentamers were observed, but there were no complexes larger than pentamers (see . These data are consistent with ␣7 subunits complexing into pentamers, similar to ␣7/5HT 3 subunit receptors (Rangwala et al, 1997) and other characterized AChRs (Karlin and Akabas, 1995). For ␣7 subunits, a large number of the multimers was present even on reducing gels or alkylated with NEM, though alkylation did disperse some of the larger multimers ( Fig.…”