Translocon pores in the endoplasmic reticulum are permeable to a neutral, polar molecule.

The pore of the translocon complex in the endoplasmic reticulum (ER) is large enough to be permeated by small molecules, but it is generally believed that permeation is prevented by a barrier at the luminal end of the pore. We tested the hypothesis that 4-methylumbelliferyl alpha-d-glucopyranoside (4MalphaG), a small, neutral dye molecule, cannot permeate an empty translocon pore by measuring its activation by an ER resident alpha-glucosidase, which is dependent on entry into the ER. The basal entry of dye into the ER of broken Chinese hamster ovary-S cells was remarkably high, and it was increased by the addition of puromycin, which purges translocon pores of nascent polypeptides, creating additional empty pores. The basal and puromycin-dependent entries of 4MalphaG were mediated by a common, salt-sensitive pathway that was partially blocked by spermine. A similar activation of 4MalphaG was observed in nystatin-perforated cells, indicating that the entry of 4MalphaG into the ER did not result simply from the loss of cytosolic factors in broken cells. We reject the hypothesis and conclude that a small, neutral molecule can permeate the empty pore of a translocon complex, and we propose that translationally inactive, ribosome-bound translocons could provide a pathway for small molecules to cross the ER membrane.

An ATP-sensitive K(+) current that regulates progression through early G1 phase of the cell cycle in MCF-7 human breast cancer cells.

Whole-cell recordings were used to identify in MCF-7 human breast cancer cells the ion current(s) required for progression through G1 phase of the cell cycle. Macroscopic current-voltage curves were fitted by the sum of three currents, including linear hyperpolarized, linear depolarized and outwardly rectifying currents. Both linear currents, but not the outwardly rectifying current, were increased by 1 microm intracellular Ca(2+) and blocked by 2 mm intracellular ATP. When tested at concentrations previously shown to inhibit proliferation by 50%, linogliride, glibenclamide and quinidine inhibited the linear hyperpolarized current, and quinidine and linogliride inhibited the linear depolarized current; none of these agents affected the outwardly rectifying current. In contrast, tetraethylammonium completely inhibited the outwardly rectifying current, but did not inhibit either linear current. Changing the bath solution to symmetric K(+) shifted the reversal potential of the linear hyperpolarized current from near the K(+) equilibrium potential (-84 mV) to -4 mV. Arrest of the cell cycle in early G1 by quinidine was associated with significantly smaller linear hyperpolarized currents, without a change in the linear depolarized or outwardly rectifying currents, but this reduction was not observed with arrest by lovastatin at a site approximately 6 hr later in G1. The linear hyperpolarized current was significantly larger in ras-transformed than in untransformed cells. We conclude that the linear hyperpolarized current is an ATP-sensitive K(+) current required for progression of MCF-7 cells through G1 phase.

Isolation of transport vesicles that deliver ion channels to the cell surface.

The squid giant axon provides a very simple preparation for the collection of bulk quantities of transport vesicles, and this greatly facilitates the physiologic study of ion channels incorporated into planar bilayers from these vesicles. However, this preparation is also limited in the repertoire of transport vesicles that can be studied, and it is not very convenient for some biochemical techniques, such as pulse-chase labeling experiments. Cultured N1E-115 cells, on the other hand, provide a preparation from which a larger repertoire of types of transport vesicles can be isolated, and many biochemical techniques can be applied in conjunction with physiologic studies. Further refinement of the techniques for isolating specific populations of vesicles from cultured cells will provide even greater insight into the role of vesicles in mediating ion channel trafficking.

Evidence for an early G1 ionic event necessary for cell cycle progression and survival in the MCF-7 human breast carcinoma cell line.

The mechanism of the G0/G1 arrest and inhibition of proliferation by quinidine, a potassium channel blocker, was investigated in a tissue culture cell line, MCF-7, derived from a human breast carcinoma. The earliest measurable effect of quinidine on the cell cycle was a decrease in the fraction of cells in S phase at 12 hr, followed by the accumulation of cells in G1/G0 phases at 30 hr. Arrest and release of the cell cycle established quinidine as a cell synchronization agent, with a site of arrest in early G1 preceding the lovastatin G1 arrest site by 5-6 hr. There was a close correspondence among the concentration-dependent arrest by quinidine in G1, depolarization of the membrane potential, and the inhibition of ATP-sensitive potassium currents, supporting a model in which hyperpolarization of the membrane potential and progression through G1 are functionally linked. Furthermore, the G1 arrest by quinidine was overcome by valinomycin, a potassium ionophore that hyperpolarized the membrane potential in the presence of quinidine. With sustained exposure of MCF-7 cells to quinidine, expression of the Ki67 antigen, a marker for cells in cycle, decreased, and apoptotic and necrotic cell death ensued. We conclude that MCF-7 cells that fail to progress through the quinidine-arrest site in G1 die.

The rate zonal separation of organelles from dilute suspensions: the problem of a large sample volume.

Transport vesicles that deliver proteins to the cell surface can be isolated by incubating cells that have been permeabilized by mechanical or chemical techniques in a high-K medium containing an ATP regenerating system. The vesicles released from permeabilized cells are, however, obtained as a very dilute suspension in the incubation solution. This presents a problem for the preparative separation of specific populations of vesicles by velocity sedimentation, because the small sample volume capacity of traditional glycerol or sucrose velocity gradients requires that the vesicles first be concentrated by sedimentation or that very small amounts of vesicles be loaded onto a gradient. We have addressed the problem of the loss of zonal resolution produced by the loading of large sample volumes, and we propose that high-viscosity Ficoll gradients can be used effectively to restore the resolution of zones when substantially larger sample volumes of dilute suspensions must be loaded onto velocity gradients.

Mitogenic signal transduction in human breast cancer cells.

1. Signal transduction pathways activated during growth of human breast cancer cells in tissue culture are reviewed. 2. Steroid hormones and growth factors stimulate similar mitogenic pathways and frequently modulate each other's activity. 3. A response common to estrogen, progestins and most polypeptide mitogens is induction of the nuclear transcription factors myc, fos and jun in early G1 phase of the cell cycle. 4. Some growth factors also stimulate cyclin D1, a regulatory protein responsible for the activation of cell cycle-dependent kinases in G1. 5. In addition, insulin, IGF-I and EGF activate tyrosine kinase receptors. 6. Several tyrosine phosphorylated proteins occur in human breast cancer cells, and include the EGF and estrogen receptors. 7. Cyclic AMP plays a critical role in breast cancer cell proliferation through the activation of protein kinase A, and it also modulates the activity of estrogen and progesterone receptors. 8. EGF is the only breast cell mitogen known to raise intracellular free calcium levels. 9. Calcium may play a dual role in breast cancer cell proliferation, activating both calmodulin-dependent processes and regulating cell membrane potential through the activation of potassium channels. 10. Potassium channel activity and cell proliferation are linked in breast cancer cells, the cell membrane potential shifting between a depolarized state in G1/G0 cells and a hyperpolarized state during S phase. 11. Activation of an ATP-sensitive potassium channel is required for breast cancer cells to undergo the G1/G0-S transition.

Changes in membrane potential during the progression of MCF-7 human mammary tumor cells through the cell cycle.

We previously reported that MCF-7 cells were arrested in the G0/G1 phase of the cell cycle by agents known to block the activity of ATP-sensitive potassium channels (Woodfork et al., 1995, J. Cell Physiol. 162:163-171). The goal of our current study was to determine if MCF-7 cells undergo changes in membrane potential during the cell cycle that might be linked to changes in K permeability. The resting membrane potentials of unsynchronized MCF-7 cells during exponential growth phase were measured using sharp glass microelectrodes, and they ranged from -58.6 mV to -2.7 mV. The distribution of membrane potentials was best fitted by the sum of four Gaussian distributions with means of -9.0 mV, -17.4 mV, -24.6 mV, and -40.4 mV. These membrane potential groups were designated D (depolarized), ID (intermediate depolarized), IH (intermediate hyperpolarized), and H (hyperpolarized), respectively. The membrane potential was sensitive to the substitution of external K and Na but not Cl. The K:Na permeability ratio increased in proportion to the negativity of the membrane potential. MCF-7 cells pharmacologically arrested in G0/G1 phase were depolarized compared to control, with cells shifted from the H and IH groups to the D group. Tamoxifen-arrested cells chased from G0/G1 into S phase by the addition of mitogenic concentrations of 17 beta-estradiol were not depolarized, and these cells were shifted from the D group back to the IH and H groups. We conclude that MCF-7 cells hyperpolarize during passage through G0/G1 and into S phase, and this hyperpolarization probably results from an increase in the relative permeability of the plasma membrane to K.

Inhibition of ATP-sensitive potassium channels causes reversible cell-cycle arrest of human breast cancer cells in tissue culture.

The purpose of this study was to determine if potassium channel activity is required for the proliferation of MCF-7 human mammary carcinoma cells. We examined the sensitivities of proliferation and progress through the cell cycle to each of nine potassium channel antagonists. Five of the potassium channel antagonists produced a concentration-dependent inhibition of cell proliferation with no evidence of cytotoxicity following a 3-day or 5-day exposure to drug. The IC50 values for these five drugs, quinidine (25 microM), glibenclamide (50 microM), linogliride (770 microM), 4-aminopyridine (1.6 mM), and tetraethylammonium (5.8 mM) were estimated from their respective concentration-response curves. Four other potassium channel blockers were tested at supra-maximal channel blocking concentrations, including charybdotoxin (200 nM), iberiotoxin (100 nM), margatoxin (10 nM), and apamin (500 nM), and they had no effect on MCF-7 cell proliferation, viability, or cell cycle distribution. Of the five drugs that inhibited proliferation, only quinidine, glibenclamide, and linogliride also affected the cell cycle distribution. Cell populations exposed to each of these drugs for 3 days showed a statistically significant accumulation in G0/G1 phase and a significant proportional reduction in S phase and G2/M phase cells. The inhibition of cell proliferation correlated significantly with the extent of cell accumulation in G0/G1 phase and the threshold concentrations for inhibition of growth and G0/G1 arrest were similar. The G0/G1 arrest produced by quinidine and glibenclamide were reversed by removing the drug, and cells released from arrest entered S phase synchronously with a lag period of approximately 24 hours. Based on the differential sensitivity of cell proliferation and cell cycle progression to the nine potassium channel antagonists, we conclude that inhibition of ATP-sensitive potassium channels in these human mammary carcinoma cells, reversibly arrests the cells in the G0/G1 phase of the cell cycle, resulting in an inhibition of cell proliferation.

Ion permeation, divalent ion block, and chemical modification of single sodium channels. Description by single- and double-occupancy rate-theory models.

Calcium ions, applied internally, externally, or symmetrically, have been used in conjunction with rate-theory modeling to explore the energy profile of the ion-conducting pore of sodium channels. The block, by extracellular and/or intracellular calcium, of sodium ion conduction through single, batrachotoxin-activated sodium channels from rat brain was studied in planar lipid bilayers. Extracellular calcium caused a reduction of inward current that was enhanced by hyperpolarization and a weaker block of outward current. Intracellular calcium reduced both outward and inward sodium current, with the block being weakly dependent on voltage and enhanced by depolarization. These results, together with the dependence of single-channel conductance on sodium concentration, and the effects of symmetrically applied calcium, were described using single- or double-occupancy, three-barrier, two-site (3B2S), or single-occupancy, 4B3S rate-theory models. There appear to be distinct outer and inner regions of the channel, easily accessed by external or internal calcium respectively, separated by a rate-limiting barrier to calcium permeation. Most of the data could be well fit by each of the models. Reducing the ion interaction energies sufficiently to allow a small but significant probability of two-ion occupancy in the 3B2S model yielded better overall fits than for either 3B2S or 4B3S models constrained to single occupancy. The outer ion-binding site of the model may represent a section of the pore in which sodium, calcium, and guanidinium toxins, such as saxitoxin or tetrodotoxin, compete. Under physiological conditions, with millimolar calcium externally, and high potassium internally, the model channels are occupied by calcium or potassium much of the time, causing a significant reduction in single-channel conductance from the value measured with sodium as the only cation species present. Sodium conductance and degree of block by external calcium are reduced by modification of single channels with the carboxyl reagent, trimethyloxonium (TMO) (Worley et al., 1986) Journal of General Physiology. 87:327-349). Elevations of only the outermost parts of the energy profiles for sodium and calcium were sufficient to account for the reductions in conductance and in efficacy of calcium block produced by TMO modification.

Ion channels in transit: voltage-gated Na and K channels in axoplasmic organelles of the squid Loligo pealei.

Ion channels that give rise to the excitable properties of the neuronal plasma membrane are synthesized, transported, and degraded in cytoplasmic organelles. To determine whether plasma membrane ion channels from these organelles could be physiologically activated, we extruded axoplasm from squid giant axons, dissociated organelles from the cytoskeletal matrix, and fused the free organelles with planar lipid bilayers. Three classes of ion channels normally associated with the plasma membrane were identified based on conductance, selectivity, and gating properties determined from steady-state single-channel recordings: (i) voltage-dependent Na channels, (ii) voltage-dependent delayed rectifier K channels, and (iii) large, voltage-independent K channels. The identity of the delayed rectifier channels was confirmed by reconstructing the time course of activation from single-channel responses to depolarizing voltage steps applied across the bilayer. These observations suggest that several classes of plasma membrane ion channels are transported in cytoplasmic organelles in physiologically active forms.

Optimizing planar lipid bilayer single-channel recordings for high resolution with rapid voltage steps.

We describe two enhancements of the planar bilayer recording method which enable low-noise recordings of single-channel currents activated by voltage steps in planar bilayers formed on apertures in partitions separating two open chambers. First, we have refined a simple and effective procedure for making small bilayer apertures (25-80 micrograms diam) in plastic cups. These apertures combine the favorable properties of very thin edges, good mechanical strength, and low stray capacitance. In addition to enabling formation of small, low-capacitance bilayers, this aperture design also minimizes the access resistance to the bilayer, thereby improving the low-noise performance. Second, we have used a patch-clamp headstage modified to provide logic-controlled switching between a high-gain (50 G omega) feedback resistor for high-resolution recording and a low-gain (50 M omega) feedback resistor for rapid charging of the bilayer capacitance. The gain is switched from high to low before a voltage step and then back to high gain 25 microseconds after the step. With digital subtraction of the residual currents produced by the gain switching and electrostrictive changes in bilayer capacitance, we can achieve a steady current baseline within 1 ms after the voltage step. These enhancements broaden the range of experimental applications for the planar bilayer method by combining the high resolution previously attained only with small bilayers formed on pipette tips with the flexibility of experimental design possible with planar bilayers in open chambers. We illustrate application of these methods with recordings of the voltage-step activation of a voltage-gated potassium channel.

Copper activates a unique inward current in molluscan neurones.

1. Reidentifiable Aplysia neurones were current and voltage clamped in vitro using standard microelectrode techniques. 2. Bath or focal application of Cu2+ at concentrations of 1-100 microM produced a rapid and reversible depolarization of the somal, but not the axonal, membrane potential. The depolarization was accompanied by an increased membrane conductance and activation of an inward current (ICu) which could not be activated by intracellular ionophoretic injection of Cu2+. 3. ICu is carried, in part, by Na+ because the reversal potential of ICu was shifted in a Nernstian fashion by decreasing the extracellular Na+ concentration. The reversal potential of ICu was not affected by removal of extracellular Ca2+ or K+. 4. ICu does not result from (1) activation of known chemically or voltage-gated Na+ conductances, (2) inhibition of the Na+-K+-ATPase or (3) a generalized increase in membrane permeability resulting from lipid peroxidation. 5. A similar inward current was activated by AgNO3 (100 microM) and HgCl2 (100 microM).

Inhibition of calcium-dependent spike after-hyperpolarization increases excitability of rabbit visceral sensory neurones.

1. Conventional intracellular recordings were made from rabbit nodose neurones in vitro. Prostaglandins D2 and E2, but not F2 alpha, produced a selective, concentration-dependent (1-100 nM) inhibition of a slow, Ca2+-dependent spike after-hyperpolarization (a.h.p.). Block of the slow a.h.p. was accompanied by an increased membrane resistance and a small (less than 10 mV) depolarization of the membrane potential. Inhibition of the slow a.h.p. produced no change in the voltage-current relationship other than the increased membrane resistance. 2. In C neurones with slow a.h.p.s, trains of brief depolarizing current pulses (2 ms duration, 0.1-10 Hz) could not elicit repetitive action potentials without failure at rates above 0.1 Hz. By contrast, C neurones without slow a.h.p.s could respond at stimulus frequencies up to 10 Hz. The frequency-dependent spike firing ability of slow a.h.p. neurones was eliminated by inhibition of the slow a.h.p. 3. Action potentials were also evoked by intrasomatic injection of paired, depolarizing current ramps (1 nA/10 ms, 0.1-5 s inter-ramp interval). For neurones without a slow a.h.p., the current threshold and number of evoked spikes were the same for both ramps, and the ramps were nearly superimposable. In neurones with a slow a.h.p., the current threshold for the first spike in the second ramp was greatly increased (300-500%) and the number of evoked spikes was reduced. Following inhibition of the slow a.h.p., the current threshold and number of evoked spikes was the same for both ramps. 4. Forskolin, a direct activator of the catalytic subunit of adenylate cyclase, also produced a concentration-dependent inhibition of the slow a.h.p., with 50% block at 30 nM. Prostaglandin D2 and forskolin produced identical enhancement of excitability in C neurones and neither substance produced any effect on C neurones that could not be attributed to inhibition of the Ca2+-dependent K+ conductance associated with the slow a.h.p. We propose that, in some visceral sensory neurones, the level of excitability is regulated by cyclic AMP-mediated control of the slow a.h.p.

A perfusion chamber for physiological studies with acutely dissociated neurons.

We describe a recording chamber that immobilizes acutely dissociated neurons on an ultra-fine mesh grid positioned above a moving stream of perfusate. This chamber is easily fabricated and has two attributes for single-electrode voltage-clamp or patch-clamp recording: (1) shallow immersion (less than 20 micron) of the neurons, and (2) stable recording with rapid perfusion rates.

Prostaglandins block a Ca2+-dependent slow spike afterhyperpolarization independent of effects on Ca2+ influx in visceral afferent neurons.

The blockade of a slow Ca2+-activated K+-dependent afterhyperpolarization (AHPs) in rabbit visceral sensory neurons by the prostaglandins, PGE1 and PGD2, was investigated to determine whether the blockade was indirectly due to a reduction in Ca2+ influx. The prostaglandins (PGs) could block the AHPs in the absence of any change in Ca2+-dependent spikes elicited in the presence of tetrodotoxin and tetraethylammonium bromide. A PG-induced decrease in Ca2+-dependent spike width observed in some neurons was temporally dissociated from the PG-induced block of the AHPs. In addition, a slow afterhyperpolarization produced by the application of the Ca2+ ionophore, A23187, was blocked by the PGs. It is concluded that a reduction in Ca2+ influx is not responsible for the PG-induced blockade of the AHPs.

Trimethyltin-induced changes in gross morphology of the hippocampus.

Acute exposure to trimethyltin (TMT) produces alterations in hippocampal morphology. The purpose of this study was to arrive at a simple method for quantitative assessment of the gross changes in morphology which could then be used as a correlate in studies of TMT toxicity. Adult Long-Evans male hooded rats were treated with a single dose of TMT chloride and sacrificed either (a) within 11 days; (b) following 30 days; or (c) 105 days following treatment. Among a variety of morphological measures explored, the easiest and most clearly dosage-related was length of the line of pyramidal cells, from CA1 through CA3c. TMT shortened this line in a dosage- and time-dependent manner. Loss of cells appeared to begin in CA3c and progress through CA3b and CA3a as a dosage and time since treatment increased. It was concluded that this measurement may provide a useful morphological correlate for physiological and behavioral studies of TMT toxicity.

Visual system dysfunction following acute trimethyltin exposure in rats.

Trimethyltin (TMT) has been shown to produce damage in the limbic system and several other brain areas. To date, damage to sensory systems has not been reported. The present study investigated the integrity of the visual system following acute exposure to TMT. Rats were chronically implanted with electrodes for recording the evoked response from either the visual cortex or optic tract following photic stimulation. Following recovery, the animals were exposed to either 0 (saline), 4, 5, 6, or 7 mg/kg trimethyltin chloride (TMT). Evoked potentials were averaged the peak-to-peak amplitudes and latencies were determined. The results indicated that exposure to TMT produced alterations in the visual evoked response. The pattern of changes suggested two effects, an alteration in retinal processing and an alteration in arousal. The manifestation of these changes was an increase in early peak latencies recorded from the visual cortex and the optic tract, a decreased amplitude recorded from the visual cortex and optic tract early peaks (all suggestive of retinal changes) and a decreased P3N3 amplitude and N3 latency recorded from the visual cortex (suggestive of increased arousal). The results demonstrate that TMT does produce alterations in sensory systems as well as in the limbic system.

Increased seizure susceptibility following trimethyltin administration in rats.

Acute treatment with trimethyltin (TMT) produces a multitude of behavioral effects including spontaneous convulsions in some animals. The present study used several different experimental seizure models to investigate seizure susceptibility in TMT-treated rats. Rats surgically implanted with electrodes in the amygdala and treated with TMT kindled more rapidly than saline-treated controls. Similarly, rats implanted with electrodes in the dorsal hippocampus kindled more rapidly than controls. Although TMT did not alter the properties of hippocampal afterdischarges, the threshold for production of after discharges was increased in both hippocampal and amygdaloid kindled rats, probably due to cell loss. TMT also increased the sensitivity of rats to pentylenetetrazol, thus suggesting that the increased seizure susceptibility was not limited to the limbic system.