Raphael Gruener

Voltage clamp of the Aplysia giant neurone: early sodium and calcium currents

1. The membrane properties of the Aplysia giant neurone were studied under controlled voltage conditions. Emphasis was placed on the early transient currents resulting from step polarizations applied while the ganglion was immersed in different test solutions.

Attenuation of channel kinetics and conductance by cholesterol: An interpretation using structural stress as a unifying concept

The ubiquity of cholesterol in cell membranes and changes in its concentration during development, aging and in various diseases suggest that it plays an important role in modulating cell function. We examined this possibility by monitoring the effects of cholesterol on the activity of the calcium-activated potassium (BK) channel reconstituted into lipid bilayers from rat brain homogenates. Increasing the cholesterol concentration to 11% of total lipid weight resulted in a 70% reduction in channel mean open time and a reduction of the open probability of the channel by 80%. Channel conductance was reduced by 7%. Cholesterol is known to change the order state and the modulus of compressibility of bilayers. These physico-chemical changes may be translated into an overall increase in the structural stress in the bilayer, and this force may be transmitted to proteins residing therein. By examining the characteristics of the BK channel as a function of temperature, in the presence and absence of cholesterol, we were able to estimate the activation energy based on Arrhenius plots of channel kinetics. Cholesterol reduced the activation energy of the BK channel by 50% for the open to closed transition. This result is consistent with an increased stress energy in the bilayer and favors the channel moving into the closed state. Taken together, these data are consistent with a model in which cholesterol induces structural stress which enhances the transition from the open to the closed state of the channel. We suggest that this is an important mechanism for regulating the activity of membrane-integral proteins and therefore membrane function, and that the concept of structural stress may be relevant to understanding the modulation of ion channel activity in cell membranes.

Changes in synaptic potential properties during acetylcholine receptor accumulation and neurospecific interactions in Xenopus nerve-muscle cell culture.

Abstract Acetylcholine receptors in the muscle cell membrane accumulate at the nerve contact area in Xenopus cell cultures. The correlation between spontaneous synaptic potential properties and extent of acetylcholine receptor accumulation was studied. Small and infrequent miniature endplate potentials were measured before acetylcholine receptor accumulation which was observed with fluorescence microscopy using tetramethylrhodamine-conjugated α-bungarotoxin. As acetylcholine receptors accumulate at the nerve contact area, these synaptic potentials become larger and their frequency increases dramatically. In nerve-contacted muscle cells where spontaneous synaptic activity could not be detected, extensive acetylcholine receptor accumulation was not found at sites of nerve contact. Furthermore, muscle cells which exhibited extensive acetylcholine receptor accumulation along the nerve always produced miniature endplate potentials. Thus acetylcholine receptor accumulation and the presence of miniature endplate potentials were strongly correlated. Noncholinergic neurons from dorsal root ganglia did not form functional synaptic contacts with muscle cells nor acetylcholine receptor accumulation along the path of contact. Furthermore, explants from tadpole spinal cord formed functional synaptic contacts with muscle cells but rarely caused AChR localization. These data are discussed in terms of developmental processes during neuromuscular junction formation.

Culture in Vector-Averaged Gravity Under Clinostat Rotation Results in Apoptosis of Osteoblastic ROS 17/2.8 Cells

Space flight experiments and studies carried out in altered gravity environments have revealed that exposure to altered gravity conditions results in (mal)adaptation of cellular function. In the present study, we used a clinostat to generate a vector‐averaged gravity environment. We then evaluated the responses of osteoblast‐like ROS 17/2.8 cells subsequent to rotation at 50 revolutions per minute (rpm) for 6–24 h. We found that the cells started to detach from the substrate between 12 h and 24 h of rotation in clinostat but not in stationary cultures or after horizontal rotation (the latter serving as a motion control for turbulence, shear forces, and vibrations). At 24 h, 35% of clinorotated cells had detached and the cells underwent apoptotic death as evidenced by DNA fragmentation analysis, terminal deoxynucleotidyl transferase‐mediated deoxyuridine triphosphate‐biotin nick end labeling (TUNEL) staining, and flow cytometry with Annexin V staining. The apoptotic death was associated with perinuclear distribution of cell‐surface integrin β1 and disorganization of actin cytoskeleton. These results suggest that vector‐averaged gravity causes apoptosis of osteoblasts by altering the organization of the cytoskeleton. We hypothesize that apoptotic death of osteoblasts might play an important role in the pathogenesis of osteoporotic bone loss as observed in actual space flights.

Lipid-ion channel interactions: Increasing phospholipid headgroup size but not ordering acyl chains alters reconstituted channel behavior

We have recently shown (Chang et al., 1995) that lipid-channel interactions, exemplified by the effects of cholesterol on the calcium-activated potassium (BK) channel, profoundly affect channel properties. The present study further explores such interactions by monitoring changes in BK channel behavior after reconstitution into bilayers where the size of phospholipid (PL) headgroups is increased and where the freedom of motion (inverse order) of fatty acid chains is incremented. Increasing the PL headgroup cross-sectional area, from that of N-meth-DOPE to that of DOPC (an increase from ca. 60 to 70 Å2), is associated with a doubling of the channel mean opentime. Channel conductance, however, was unaffected. Increasing the order of the fatty acid chains, from that of DOPE to POPE and to that of DEPE, had no significant effect on channel properties (at 22°C). We interpret the changes reported here to reflect lipid-protein interactions through the induction of structural stress related to the headgroup structures of phospholipids.

Hyperthyroid myopathy. Intracellular electrophysiological measurements in biopsied human intercostal muscle.

Morphological and electrophysiological studies were performed on intercostal muscle biopsies from 2 thyrotoxic patients. The diseased fibers had numerous areas of subsarcolemmal glyogen accumulations and abnormal membranous projections. Both Type I and Type II muscle fibers were atrophied. Diseased fibers were substantially depolarized and when artifically hyperpolarized showed earlier inactivation of the sodium conductance as a function of membrane potential, and a critical depolarization potential more depolarized than in normal fibers. When stimulated at 20 pulses/sec, or faster, the diseased fibers could not generate normal action potentials due to membrane depolarization and the appearance of a marked after-hyperpolarization. Muscle weakness associated with hyperthyroidism is attributed to the reduced membrane excitability.

Microarray Analysis of Spaceflown Murine Thymus Tissue Reveals Changes in Gene Expression Regulating Stress and Glucocorticoid Receptors

The detrimental effects of spaceflight and simulated microgravity on the immune system have been extensively documented. We report here microarray gene expression analysis, in concert with quantitative RT‐PCR, in young adult C57BL/6NTac mice at 8 weeks of age after exposure to spaceflight aboard the space shuttle (STS‐118) for a period of 13 days. Upon conclusion of the mission, thymus lobes were extracted from space flown mice (FLT) as well as age‐ and sex‐matched ground control mice similarly housed in animal enclosure modules (AEM). mRNA was extracted and an automated array analysis for gene expression was performed. Examination of the microarray data revealed 970 individual probes that had a 1.5‐fold or greater change. When these data were averaged (n = 4), we identified 12 genes that were significantly up‐ or down‐regulated by at least 1.5‐fold after spaceflight (P ≤ 0.05). The genes that significantly differed from the AEM controls and that were also confirmed via QRT‐PCR were as follows: Rbm3 (up‐regulated) and Hsph110, Hsp90aa1, Cxcl10, Stip1, Fkbp4 (down‐regulated). QRT‐PCR confirmed the microarray results and demonstrated additional gene expression alteration in other T cell related genes, including: Ctla‐4, IFN‐α2a (up‐regulated) and CD44 (down‐regulated). Together, these data demonstrate that spaceflight induces significant changes in the thymic mRNA expression of genes that regulate stress, glucocorticoid receptor metabolism, and T cell signaling activity. These data explain, in part, the reported systemic compromise of the immune system after exposure to the microgravity of space. J. Cell. Biochem. 110: 372–381, 2010.

Excitability modulation by taurine

The presence of taurine, a non-essential amino acid, in nerve and muscle has been previously associated with inhibition of activity in the central nervous system, with the etiology of epileptogenic foci, and with the muscle weakness of muscular dystrophy. We present here data showing a small and probably insignificant effect of taurine on neuromuscular transmission per se, but significant hyperpolarization of the membrane potential in both taurine-incubated and taurine-loaded muscles. In addition, we found that taurine reduces the time course of the muscle action potential. The results are interpreted in terms of neuromuscular transmission and excitation-contraction coupling consequent to these phenomena. This interpretation is compatable with the hypothesis that taurine is involved in the genesis of muscular dystrophy where the membrane potential is depolarized. Our results and interpretation can also explain the anti-arrhythmic action of taurine on cardiac muscle.

Halothane-induced changes in acetylcholine receptor channel kinetics are attenuated by cholesterol

The single-channel recording technique was used to investigate the role of membrane lipids in the action of general anesthetics on ion channels. We examined the effects of halothane on acetylcholine receptor channels in Xenopus laevis myocytes in which the plasma membrane cholesterol level had been changed by pretreatment with cholesterol-rich or cholesterol-free liposomes. We found that the alteration in acetylcholine receptor channel kinetics, elicited in the presence of clinically-relevant concentrations of halothane, is attenuated when membrane cholesterol is increased and enhanced when membrane cholesterol concentration is decreased. These findings support the idea that general anesthetics interact with synaptic receptor channels indirectly through the lipid domains in which these synaptic proteins are embedded.

Distribution and density of α-bungarotoxin binding sites on innervated and noninnervated Xenopus muscle cells in culture

Abstract The distribution and density of α-bungarotoxin (α-BT) binding sites on Xenopus muscle cells in culture by autoradiography using 125 I-α-BT were examined. In muscle cells grown alone α-BT binding sites were fairly uniformly distributed over the entire surface with a mean density of 104/μm 2 (background density). Occasionally, spots of higher density were observed (“hot spots”) where the mean density was 890/μm 2 . The addition of neural tube cells did not change the background density. Similarly in the majority of cases medium contained with neural tube cells did not affect the density of α-BT binding sites. Previous findings that the background acetylcholine sensitivity of muscle cells increased in the presence of neural tube cells (by approximately 50%) or in conditioned medium (by approximately 70%), therefore, are not likely due primarily to an increase in the acetylcholine receptor (AChR) density. In cocultures of nerve and muscle cells regions of high α-BT binding sites were occasionally associated with the path of neurites. In such regions the density of α-BT binding sites was estimated to be approximately 1000/μm 2 . However, even in these cells the density at non-nerve contacted regions was not different from that in muscle cells cultured alone. Whether the increase in AChR density at the junctional area is sufficient to explain a previous observation of a fivefold increase in the amplitude of spontaneous synaptic potentials during the process of AChR accumulation is discussed.