Joe Henry Steinbach

The Extracellular Patch Clamp: A Method for Resolving Currents through Individual Open Channels in Biological Membranes

The current contributions of individual ionic channels can be measured by electrically isolating a small patch of membrane. To do this, the tip of a small pipette is brought into close contact with an enzymatically cleaned membrane of a hypersensitive amphibian or mammalian muscle fiber. Current flowing through the pipette is measured. If the pipette contains cholinergic agonist at μ-molar concentrations, square pulse current waveforms can be observed which represent the activation of individual acetylcholine-receptor channels. The square pulses have amplitudes of 1 to 3 pA and durations of 10–100 ms.In order to obtain the necessary resolution, a delicate compromise had to be found between different experimental parameters. Pipettes with 1–3 μm internal diameter and a steep final taper had to be used, extensive enzyme treatment was necessary, and conditions had to be found in which channels open at a relatively low frequency.

The distribution of α‐bungarotoxin binding sites on mammalian skeletal muscle developing in vivo

1. The distribution of α‐bungarotoxin binding sites on embryonic and neonatal rat skeletal muscle fibres was determined by autoradiography. Most of the bungarotoxin binding could be inhibited by curare. This observation, together with the spatial distribution of toxin‐binding sites, indicates that the distribution of bound toxin reflects that of acetylcholine (ACh) receptors on these developing muscle cells.

Activation of acetylcholine receptors on clonal mammalian BC3H-1 cells by low concentrations of agonist.

The patch‐clamp technique was used to examine the activation of single acetylcholine receptor channels of clonal BC3H‐1 mouse muscle cells. Single‐channel currents were activated by low concentrations of the strong agonists acetylcholine (ACh, 50‐100 nM), carbamylcholine (1‐2 microM), and suberyldicholine (30‐50 nM). At low agonist concentrations channel openings occur as isolated short‐duration openings and as bursts of longer duration openings separated by brief closed periods. Two distinct types of brief closed periods separate long duration openings: brief closures (mean duration, 50 microseconds) and intermediate closures (mean duration, 0.5‐1.0 ms). The kinetic properties of intermediate closures depend on the agonist, suggesting that they reflect receptor reopening from the closed state leading to the open state. Properties of brief closures, in contrast, are independent of the agonist, indicating that they result from an additional closed state leading away from the pathway producing the open state. A receptor activation scheme is proposed which accounts for the observed closed states, and transition rate estimates are presented for steps within the proposed scheme. The channel opening rate, beta, differs several‐fold for the agonists studied (200‐1400 s‐1) and is comparable to the dissociation rate, k‐2 (900 s‐1). The dissociation rate is similar for the three agonists studied. The channel closing rate, alpha, is much slower than the opening rate (20‐60 s‐1). The probability is high that a doubly liganded channel is in the open state and depends on the agonist (0.75‐0.97). Beta increases and alpha decreases at more negative membrane potentials, whereas k‐2 shows little potential dependence.

Developmental changes in acetylcholine receptor aggregates at rat skeletal neuromuscular junctions

The development of acetylcholine (ACh) receptor aggregates at the neuromuscular junction was studied in rat sternomastoid muscles. The first junctional clusters of ACh receptors were loose aggregates of small receptor patches (15 1/2 to 16 1/2 days of gestation). These clusters coalesced to more compact but simple plaques (18 days of gestation to 3 days postnatal). During the first 2 weeks postnatal several changes occurred: the receptor plaque was modified to the adult junctional receptor distribution, multiple innervation was eliminated, and extrajunctional ACh receptors were lost. At the time of birth the receptors in the junctional receptor plaque were already degraded more slowly than were extrajunctional receptors. The junction increased in length most rapidly during the period when the muscle increased most rapidly in mass, from 15 to 100 days postnatal. It is concluded that the junction goes through several stages during development.

Reversible loss of acetylcholine receptor clusters at the developing rat neuromuscular junction.

Exposure of sternomastoid muscles excised from 16-day embryonic rats to medium depleted of Ca2+ or containing high concentrations of KCl leads to extensive loss of aggregates of acetylcholine receptors newly formed at the motor end plate region. Upon restoration of Ca2+ or removal of excess KCl, receptor accumulations reappear in the central regions of about one-third of the muscle fibers. This susceptibility of junctional AChR aggregates lasts only a short while during development of the neuromuscular junction. By the time of birth, end plate receptor aggregates have become resistant to these treatments.

Channel open time of acetylcholine receptors on Xenopus muscle cells in dissociated cell culture

The kinetics of acetylcholine (ACh) receptor channels on cultured myotomal muscle cells from Xenopus embryos were studied by analyzing focally recorded membrane currents. The mean open time for receptor channels on embryonic muscle cells grown in dissociated cell cultures showed a time-dependent decrease similar to that seen in vivo. The changes in power density spectra are consistent with the hypothesis that the decrease results from the appearance of a class of ACh receptor with a short mean channel open time (0.7 msec) and a decrease in the proportion of receptors with a long mean channel open time (3 msec). The addition of dissociated neural tube cells to muscle cell cultures resulted in an unexpected increase in mean channel open time for ACh receptors in both synaptic and nonsynaptic regions. These studies demonstrate that ACh receptor function may be altered in cultured muscle cells.

FACTORS AFFECTING THE SUSCEPTIBILITY OF DIFFERENT STRAINS OF MICE TO EXPERIMENTAL MY ASTHENIA GRAVIS

Mice immunized with purified AChR (T. californica) invariably form anti-AChR antibodies and often develop a condition of extreme muscular weakness and flaccid paralysis. Pharmacological, physiological, and ultrastructural studies indicate that the pathophysiology of EMG in the mouse closely resembles that of human MG. The single episode of muscular weakness typically found in mouse EMG differs from the acute phase of rat EMG in that macrophages and other phagocytes do not appear to play an active role in the destruction of the neuromuscular junction. The frequency of paralysis in mice immunized with AChR is highly strain dependent and is not attributable to polymorphisms with respect to susceptibility to cholinergic blockade. The incidence of paralysis does not correlate with the magnitude of the humoral response to either T. californica or mouse AChR. Because both paralyzed and nonparalyzed mice form antibodies which are able to increase the rate of both junctional and extrajunctional AChR degradation, the mere presence of antibodies reactive with cell surface antigenic determinants of AChR is not sufficient for the induction of paralysis. While it is still possible that antibody-induced degradation of AChR may be necessary for the induction of paralysis, these studies rule out the possibility that antigenic modulation of AChR is sufficient to account for the induction of paralysis in mouse EMG. In the present studies alleles of the two loci were identified which significantly effect the probability with which mice immunized with AChR can be expected to become paralyzed, the MHC and the IgCH region. Because one genotype, H-2b, Ig-1b segregated with high susceptibility to EMG in four strains derived from three dissimilar backgrounds, these studies strongly suggest that susceptibility to the development of paralysis is a heritable trait determined by regions of the mouse genome which regulate immune responsiveness.

Collagenase digestion alters the organization and turnover of junctional acetylcholine receptors

Acetylcholine receptors at the vertebrate neuromuscular junction are highly organized and metabolically very stable. We report here that digestion of adult rat skeletal muscle with collagenase disorganizes junctional receptors and increases their turnover rate.