Spontaneous changes in acetylcholine receptor and calcium leakage activity in cell-attached patches from cultured dystrophic myotubes

Abstract

Calcium leakage activity (CLA) was recorded in association with acetylcholine receptor (AChR) activity in cell-attached patches from cultured nondystrophic and dystrophic (mdx) myotubes. Cell-attached recordings from dystrophic myotubes exhibited spontaneous transitions in the activity pattern that were characterized by an instability of AChR function and a decrease in the frequency of AChR events. Recordings from nondystrophic myotubes could be maintained for similar time periods without observing any consistent changes in the distribution of CLA and AChR events, thus indicating that the conditions of the experiment were not conducive to developing AChR instability or desensitization in nondystrophic myotubes. In dystrophic myotubes, the decrease in AChR event frequency was associated with an increase in small-conductance events which had the characteristics of CLA, and the subsequent acquisition of an inside-out patch appeared to restore the AChR activity. Examination of baseline current-voltage relationships indicated that dystrophic and nondystrophic patches exhibited the same general pattern of seal maturation with temporal increases in the total-patch circuit resistance. These results are discussed in relation to the AChR aggregation hypothesis, which proposes that the absence of dystrophin leads to abnormal AChR-cytoskeletal interactions and CLA that can be reversed by removing the influence of intracellular signal transduction enzymes that aggregate and stabilize AChR clusters.

Adult dystrophic (mdx) endplates exhibit reduced quantal size and enhanced quantal variation

“…Since there is no evidence for a functional or morphological defect at mdx nerve terminals or for any alterations in ACh esterase (AChE) activity [19,21], the reduction in the mean quantal response indicates a reduction in the mean number of functional AChRs aggregated in apposition to presynaptic release sites. This reduction could be produced by the presence of AChR aggregates of low density apposed to a select proportion of individual presynaptic release sites, to a uniform distribution of AChR aggregates between release sites in which a proportion of the AChRs at each site have a stable reduced conductance, and/or to temporal variations in ACh affinity or single-channel conductance [5] at AChR aggregates apposed to presynaptic release sites. The distinct MEPP mean/variance relation seen at mature mdx endplates (Figs.…”

“…Cell-attached patches from cultured mdx myotubes also show significantly elevated Ca 2+ leakage event frequencies in the presence of ACh compared with patches from non-dystrophic myotubes, and the population of patches exhibits an inverse relationship between Ca 2+ leakage activity (CLA) and AChR event frequency [3]. Acquisition of an inside-out patch increases AChR activity and reduces CLA in mdx membranes [3,5,7], thus suggesting that intracellular modulators may affect AChR function in mdx myotubes. These results indicate that AChR single-channel function is unstable in cellattached patches from cultured mdx myotubes [5].…”

“…Acquisition of an inside-out patch increases AChR activity and reduces CLA in mdx membranes [3,5,7], thus suggesting that intracellular modulators may affect AChR function in mdx myotubes. These results indicate that AChR single-channel function is unstable in cellattached patches from cultured mdx myotubes [5].…”

“…In cultured mdx myotubes, long-term (>5 min), cellattached recordings exhibit spontaneous reductions in the frequency of AChR openings and concomitant increases in the frequency of lower conductance Ca 2+ leakage events [5]. Cell-attached patches from cultured mdx myotubes also show significantly elevated Ca 2+ leakage event frequencies in the presence of ACh compared with patches from non-dystrophic myotubes, and the population of patches exhibits an inverse relationship between Ca 2+ leakage activity (CLA) and AChR event frequency [3].…”

“…However, since the AChR instability is observed in cell-attached patches it is possible that these effects had been induced by membrane stretch or other physical forces associated with the formation and/or maintenance of a cell-attached patch [5]. It was therefore considered necessary to examine AChR function at intact, unperturbed, mdx endplates.…”