Lambert-Eaton syndrome IgG inhibits transmitter release via P/Q Ca2+ channels

Abstract

Objective: To determine whether immunoglobulin G (IgG) from patients with Lambert-Eaton myasthenic syndrome (LEMS) decreases action potential–evoked synaptic vesicle exocytosis,and whether the effect is mediated by P/Q-type voltage-gated calcium channels (VGCCs).

Methods: IgG was obtained from 4 patients with LEMS (3 males, 1 female), including 2 patients with lung malignancy. Antibodies against P/Q-type VGCCs were detected in all 4 patients, and against N-type VGCCs in 2. We incubated neuronal cultures with LEMS IgG and determined the size of the total recycling pool of synaptic vesicles and the rate of action potential–evoked exocytosis using fluorescence imaging of the amphiphilic dye SynaptoRed C1. Pooled IgG from healthy volunteers was used as a control. We repeated the experiments on synapses lacking P/Q-type calcium channels from a Cacna1a knockout mouse to determine whether these channels account for the pathogenic effect of LEMS IgG.

Results: LEMS IgG had no effect on the total recycling pool size but significantly reduced the rate of action potential–evoked synaptic exocytosis in wild-type neurons when compared with neurons treated with control IgG. In contrast, LEMS IgG had no effect on the rate of synaptic vesicle exocytosis in neurons lacking P/Q-type channels.

Conclusions: These data provide direct evidence that LEMS IgG inhibits neurotransmitter release by acting on P/Q-type VGCCs.

Figure 1. Fluorescence measurements of evoked vesicular release in rat cultures after incubation in control or LEMS IgG

(A) Experimental protocol showing the sequence of SRC1 incubation, stimulation to load boutons, to evoke exocytosis, and to achieve complete destaining, and fluorescence imaging. (B, C) Representative SRC1 imaging experiments in cultures treated with control (B) or with LEMS IgG (C). Fluorescence microscopy images (top) show gradual decrease of fluorescence at successive time points as indicated during the experiment. Fluorescence time courses in 2 pairs of representative boutons (arrows) are shown below in relative fluorescence units (RFU). Spontaneous and evoked destaining rates were fitted with mono-exponential curves. The specific action potential–dependent rate of destaining k_AP was calculated as k_AP = k_EV − k_SP. Scale bars: 5 μm. AP = action potential; IgG = immunoglobulin G; LEMS = Lambert-Eaton myasthenic syndrome; SRC1 = SynaptoRed C1.

Figure 2. LEMS IgG reduces evoked exocytosis but not the total recycling pool size

Effects of LEMS and control IgG on k_AP (A) and relative TRP size (B). Left panels show cumulative distributions of mean k_AP and TRP size values obtained in individual experiments (average of 10–50 boutons in each experiment). Data derived with samples obtained from each of 4 patients with LEMS are shown as thin colored lines. LEMS samples 1–4 are color-coded as in the legend, and were used in 3, 9, 8, and 6 experiments, respectively. Thick red line, pooled LEMS IgG data; thick black line, control IgG data. Right panels show the mean (±SEM) values for the pooled data (LEMS IgG n = 26 experiments, control IgG n = 21 experiments, *p < 0.05). IgG = immunoglobulin G; LEMS = Lambert-Eaton myasthenic syndrome; ns = nonsignificant; RFU = relative fluorescence units; TRP = total recycling pool.

Figure 3. P/Q-type channels are required for inhibition of vesicular release by LEMS IgG

Effects of LEMS and control IgG on k_AP (A) and relative TRP size (B) in WT and Cacna1a^+/− (KO) neurons. Left panels show cumulative distributions of mean k_AP and TRP size values obtained in individual experiments. Right panels show the mean (±SEM) values for the pooled data. Data are from 602 boutons in 15 experiments (WT, control, black line), 138 boutons in 5 experiments (WT, LEMS, gray), 303 boutons in 15 experiments (KO, control, red), and 175 boutons in 6 experiments (KO, LEMS, pink). **p < 0.01. IgG = immunoglobulin G; KO = knockout; LEMS = Lambert-Eaton myasthenic syndrome; ns = nonsignificant; RFU = relative fluorescence units; TRP = total recycling pool; WT = wild-type.