Faculty Bibliography

G292 osteoblastic cells were cultured in dishes made with a flexible base of polytetrafluoroethylene (PTFE) and stretched ( approximately 1% strain level) continuously for 48 hours. Patch-clamp recording techniques were then used to monitor single channel currents of mechanosensitive ion channels in these cells. To stimulate mechanosensitive channels, we applied suction to the membrane, expressed as -cm Hg, directly through the patch pipette. GigaOhm seals were obtained on a total of 33 osteoblasts that contained a high-conductance ( approximately 180 pS) mechanosensitive channel, all in the cell attached configuration. Of these, 18 were obtained from cells that had been stretched for either 1 (n = 6), 24 (n = 4), or 48 (n = 8) hours, and 15 were obtained in control (nonstretched) cells at either 1 (n = 2), 24 (n = 5), or 48 (n = 8) hours. For unstrained cells, applied pressures ranging from -1 to -5 cm Hg increased the probability of channel opening (Popen) from 0.05 +/- 0. 01 (mean + SEM) to 0.12 +/- 0.07. By contrast, for the same values of applied pressure in stretched cells, Popen ranged from 0.06 +/- 0. 01 to 0.49 +/- 0.15. Our results suggest that intrinsic properties of mechanosensitive ion channels in the G292 osteoblastic cell may be modulated by continuous mechanical loading of the cell itself.

Kinetics of voltage-gated ionic channels fundamentally reflect the response of the channels to local electric fields. In this report cell-attached patch-clamp studies reveal that the voltage-dependent activation rate of sodium channels residing in the growth cone membrane differs from that of soma sodium channels in differentiating N1E-115 neuroblastoma cells. Because other electrophysiological properties of these channels do not differ, this finding may be a reflection of the difference in intramembrane electric field in these two regions of the cell. This represents a new mechanism for channels to attain a range of activities both within and between cells

Clinical studies have established that NaF increases mineral content in bone, although the cellular mechanisms underlying its osteoinductive effects remain unclear. Because metabolic effects of fluoride have been linked to ion flux and alterations in membrane potential, we used patch-clamp recording techniques to examine the electrophysiological response of osteoblastic cells to NaF. In these experiments, we show that NaF increased the amplitude and P(open) of a 73 pS potassium-selective ion channel. The effect of NaF depended on extracellular Ca2+ and could be blocked by a combination of calcium-channel blocking agents, suggesting that potentiation of channel activity was dependent on external calcium. Because all patches were in the cell-attached configuration, the effect of NaF was presumable indirect. Although the underlying cellular mechanisms remain unclear, our findings suggest that activity of calcium and/or potassium-selective channels via second messenger cascades may mediate many of the early events involved in the response of bone cells to inorganic fluoride.

Intradental, i.e. pulpal, cells may play an important part in sensory transduction in teeth, although the cellular mechanisms and the identity of the specific cell types involved are still unclear. Because the majority of cells in dental pulp are derived from neural crest, it seemed likely that these might have the membrane properties of other neural-derived cells found in the peripheral or central nervous system. The patch-clamp recording technique was used to show that cells in explant cultures from human dental pulp contain a voltage-gated, tetrodotoxin-sensitive inward current. Mean activation potential of the current was -42 +/- 2.5 mV and the voltage at half-inactivation was -79.4 +/- 5.3 mV, suggesting a neural-like sodium conductance. In addition, these cells were immunoreactive to glial acidic fibrillary protein, growth-associated protein (GAP-43), and vimentin, further suggesting that dental pulp contains a population of cells with membrane properties similar to neuronal satellite cells. These cells may contribute, either directly or indirectly, to somatosensation in teeth.

The neurodegenerative pathology observed in Alzheimer's Disease (AD) has been partially attributed to the neurotoxic effects of the amyloid beta-peptide (A beta P), although the mechanisms underlying this neurotoxicity are unknown. Since A beta P is capable of forming cation channels in lipid bilayers, it is possible that the neurotoxic effects on neurons may be mediated by a cation flux. We have used patch-clamp recording techniques to study the effects of A beta P on cation currents in differentiated mouse N1E-115 neuroblastoma cells. In whole-cell recordings, incubation of cells with A beta P for 24 h significantly increased the median peak inward current from -201.8 pA to -352.0 pA, and shifted the voltage at peak current (Vpeak) and that of current activation (Vact) towards more positive potentials. For untreated cells, median Vpeak was 1.7 mV and Vact was -28.9 mV, vs. 10.5 mV and -24.7 mV in A beta P-treated cells. Incubation with the reverse sequence A beta P(40-1) or A beta P(25-35) did not produce significant changes in the amplitude or kinetic behavior of the inward current. At the single channel level, A beta P added to the pipette increased the open probability of cation-conducting ion channels. As determined by cell viability counts, both A beta P(1-40) and the A beta P(25-35) fragment had neurotoxic effects; within 24 h, addition of A beta P reduced the number of viable cells by more than 50%. It is suggested that the neurotoxic effects of A beta P may be mediated by its ability to form cation channels de novo and/or alter the activity of cation channels already present in the cell membrane.

Patch-clamp recording methods were used to monitor ion currents in tissue-cultured cells derived from human dental pulp. Recordings were made in excised, outside-in or whole-cell patches. In single-channel experiments, the majority of patches contained a high-conductance (approx. 140-180 pS) K(+)-selective ion channel. The probability of the channel being in an open state was dependent on membrane potential, internal calcium and negative pressure applied to the cell membrane. Whole-cell recordings were consistent with these findings; in response to step-wise depolarizations of the cell membrane, most displayed a family of outwardly rectifying, barium-sensitive currents. In addition, a number of patches contained a second class of potassium channel of intermediate (approx. 85-100 pS) conductance, which was largely voltage insensitive and independent of calcium concentration. These results suggest that pulp cells contain a high-conductance potassium channel which probably underlies the outwardly rectifying current found at the whole-cell level. Further, the existence of mechanosensitive channels in these cells raises the possibility that the response to mechanical perturbation of dental pulp may be mediated, in part, by direct effects on odontogenic cells.

A high-conductance K(+)-selective ion channel was studied in excised membrane patches from human G292 osteoblast-like osteosarcoma cells. Channel conductance averaged approximately 170 pS in symmetric solutions of 153 mM KCl, and approximately 135 pS when the pipette was filled with standard saline (150 mM NaCl). The probability of the channel being in an open state (Popen) increased with membrane potential, internal calcium, and applied negative pressure. At pCa7, channel activity was observed at membrane potentials greater than approximately 60 mV, while at pCa3, channel activity was seen at approximately 10 mV. Likewise, in the absence of applied pressure, channel openings were rare (Popen = 0.02), whereas with -3 cm Hg applied pressure, Popen increased to approximately 0.40. In each case, i.e., voltage, calcium concentration, and pressure, the increase in Popen resulted from a decrease in the duration of long-closed (interburst) intervals and an increase in the duration of long-open (burst) intervals. Whole-cell responses were consistent with these findings. Hypotonic shock produced an increase in the amplitude and conductance of the outward macroscopic current and a decrease in its rise time, and both single-channel and whole-cell currents were blocked by barium. It is suggested that the voltage-gated, calcium dependent maxi-K+ channel in G292 osteoblastic cells is sensitive to membrane stretch and may be directly involved in osmoregulation of these cells. Further, stretch sensitivity of the maxi-K+ channel in osteotrophic cells may represent an adaptation to stresses associated with mechanical loading of mineralized tissues.

Neurons were recorded in the superficial layers of the superior colliculus in anesthetized monkeys. As classically described, cells were non-selective for target direction and speed when the target moved through an empty visual field. However, these same cells were sensitive to target direction and speed relative to a textured moving background. The target's response was suppressed when its direction and speed were similar to that of the background, irrespective of the absolute direction of background movement.