binding studies were performed in 96-well plates, using 0.05-2 nmol/L of 125 I-โฃ-btx (25ยฐC, 60 min). Counts per minute were analyzed by nonlinear least squares regression (EBDA-LIGAND program, Rhodbard and McPherson, version 3.0) to determine equilibrium dissociation constant (K d ) and density (B max ) of binding sites.
While control lymphocytes bound โฃ-btx with a K d range of 0.6 to 1.6 nmol/L and density of 35 to 125 fmol per million cells, no binding was observed in 4 of 5 CM patients (Fig. 1). Baseline lymphocyte 3 H-thymidine uptake was similar in CM patients and controls; it was suppressed by 10 -6 and 10 -9 mol/L of CC. Proliferative response to Con A, observed in all subjects, was inhibited by 10 -6 mol/L CC but not 10 -9 mol/L. D-tc abolished these effects of CC.
Our results suggest decreased โฃ-btx binding in CM patients' lymphocytes, indicating a possible defect on lymphocyte AChR in addition to muscle AChR. In fact, AChR mutations resulting in decreased expression of AChR on cell membrane were found in 3 of these 4 CM patients after this study. 6 Functional assays suggest both nicotinic and muscarinic AChR in lymphocytes. [7][8][9] The nicotinic action of CC in this study was similar in CM patients and controls, suggesting normal lymphocyte AChR function, even in CM cases with scarce btx binding sites. This implies that binding studies may not demonstrate all receptors for CC or that a small number of AChR, below the level of detection by binding studies, is enough to respond to the agonist CC.
Our preliminary study involving a small number of subjects suggests AChR status can be evaluated on tissues other than muscle-and lymphocytes may provide an easily accessible source. Lymphocyte response to cholinergic agents appears to be normal in CM; however, these results need to be confirmed in larger groups.