William F. Wonderlin

Potassium Channels, Proliferation and G1 Progression

Potassium channels are the most ubiquitous and diverse family of plasma membrane ion channels, and this is reflected in a large variety of essential roles they perform in different cells. Voltage-gated K channels modulate the excitability of excitable cells, and K channels gated by intracellular ligands, such as calcium or ATP, provide a functional link between the physiological properties of the plasma membrane and the activity of intracellular metabolic pathways. There is now substantial evidence that drugs which block K channels also inhibit the proliferation of many types of cells, but the cellular mechanism(s) by which the level of K channel activity might be related to proliferation remains unclear. A particularly intriguing possibility is that the opening, or activation, of K channels might be required for the passage of cells through a specific stage in their cell cycle; this would provide a fundamental link between physiological and biochemical signaling pathways which regulate progression through the cell cycle (Fig. 1). The role of K channels in mitogenesis and proliferation has been previously reviewed [20,24,60]. The focus of the present review is the hypothesis that the activation of K channels is required for cells to progress through the G1 phase of the cell cycle. We will examine first the evidence supporting this hypothesis, and then we will discuss the processes or events within G1 phase that are most likely to require the activation of K channels. Identification of these critical events is important because their dependence on the activation of ion channels in the plasma membrane would represent a novel type of regulatory checkpoint, compared to other checkpoints previously identified within the G1 phase of the cell cycle [66].

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.

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.

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 others 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.

Resistance training increases heat shock protein levels in skeletal muscle of young and old rats

Heat shock proteins (HSP) HSP72, HSC70 and HSP25 protein levels and mRNA levels of HSP72 genes (Hsp72-1, Hsp72-2, Hsp72-3) and HSC70 were examined in tibialis anterior muscles from young and old rats following 4.5 weeks of heavy resistance exercise. Young (3 months) (n=10) and old (30 months) (n=9) rats were subjected to 14 sessions of electrically evoked resistance training using stretch-shortening contractions of the left limb that activated the dorsiflexor muscle group, including the tibialis anterior muscle, while the right side served as the intra-animal control. Muscle wet weight of the left tibialis anterior increased by 15.6% in young animals compared to the untrained right side, while the aged rats demonstrated no significant hypertrophy based on muscle wet weight. There were no differences in mRNA expression between the control and experimental muscles in either the old or the young animals for any of the four genes examined. On the other hand, HSP72 levels as determined by Western blots were significantly (p<0.01) higher (968.8 and 409.1%) in the trained as compared to the contralateral control muscle in young and old animals, respectively. HSP25 expression was increased significantly (p<0.01) by training in muscles of young rats (943.1%) and old rats (420.3%). Moreover, there was no training by age interaction for HSP72, while a significant age and training by age effects were found in muscles for HSP25. There was no change in HSC70 protein expression in response to the training intervention in either age group. SOD-1 enzyme level increased by 66.6% in the trained muscles of the young rats, while this enzyme was 33% lower in trained muscles compared to the untrained control side in old rats. Moreover, a significant (p<0.05) training by age interaction was found for SOD-1 enzyme levels. This study suggests that fast contracting muscles in young and old animals are capable of increasing HSP expression in response to high intensity contractile stress. Furthermore, the data are consistent with the hypothesis that higher levels of oxidative stress in muscles of old animals limit HSP levels and/or function in response to high intensity contractile stress.

A KLF4–miRNA-206 Autoregulatory Feedback Loop Can Promote or Inhibit Protein Translation Depending upon Cell Context

ABSTRACT Krüppel-like factor 4 (KLF4), a transcription factor that regulates cell fate in a context-dependent fashion, is normally induced upon growth arrest or differentiation. In many cancer cells there is dysregulation, with increased expression in proliferating cells. To identify sequence elements that mediate KLF4 suppression in normal epithelial cells, we utilized a luciferase reporter and RK3E cells, which undergo a proliferation-differentiation switch to form an epithelial sheet. A translational control element (TCE) within the KLF4 3′-untranslated region interacted with microRNAs (miRs) 206 and 344-1 to promote or inhibit KLF4 expression, respectively, in proliferating epithelial cells. Overall, the TCE suppressed expression in proliferating primary human mammary epithelial cells, but this suppressive effect was attenuated in immortalized mammary epithelial MCF10A cells, in which Dicer1 and miR-206 promoted KLF4 expression and TCE reporter activity. In contrast to MCF10A cells, in breast cancer cells the activity of miR-206 was switched, and it repressed KLF4 expression and TCE reporter activity. As miR-206 levels were KLF4 dependent, the results identify a KLF4–miR-206 feedback pathway that oppositely affects protein translation in normal cells and cancer cells. In addition, the results indicate that two distinct miRs can have opposite and competing effects on translation in proliferating cells.

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

Abstract. 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 μm intracellular Ca2+ 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 ≈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.

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.

The Permeability of the Endoplasmic Reticulum Is Dynamically Coupled to Protein Synthesis

Proteins synthesized by the rough endoplasmic reticulum (RER) co-translationally cross the membrane through the pore of a ribosome-bound translocon (RBT) complex. Although this pore is also permeable to small molecules, it is generally thought that barriers to their permeation prevent the cyclical process of protein translation from affecting the permeability of the RER. We tested this hypothesis by culturing Chinese hamster ovary-S cells with inhibitors of protein translation that affect the occupancy of RBTs by nascent proteins and then permeabilizing the plasma membrane and measuring the permeability of the RER to a small molecule, 4-methyl-umbelliferyl-α-d-glucopyranoside (4-MαG). The premature or normal release of nascent proteins by puromycin or pactamycin, respectively, increased the permeability of the RER to 4-MαG by 20–30%. In contrast, inhibition of elongation and the release of nascent proteins by cycloheximide did not increase the permeability, but it prevented the increase in permeability by pactamycin. We conclude that the permeability of the RER is coupled to protein translation by a simple gating mechanism whereby a nascent protein blocks the pore of a RBT during translation, but after release of the nascent protein the pore is permeable to small molecules as long as an empty ribosome remains bound to the translocon.

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).