Is there a role for voltage-gated Na+ channels in the aggressiveness of breast cancer?

Breast cancer is the most common cancer among women and its metastatic potential is responsible for numerous deaths. Thus, the need to find new targets for improving treatment, and even finding the cure, becomes increasingly greater. Ion channels are known to participate in several physiological functions, such as muscle contraction, cell volume regulation, immune response and cell proliferation. In breast cancer, different types of ion channels have been associated with tumorigenesis. Recently, voltage-gated Na+ channels (VGSC) have been implicated in the processes that lead to increased tumor aggressiveness. To explain this relationship, different theories, associated with pH changes, gene expression and intracellular Ca2+, have been proposed in an attempt to better understand the role of these ion channels in breast cancer. However, these theories are having difficulty being accepted because most of the findings are contrary to the present scientific knowledge. Several studies have shown that VGSC are related to different types of cancer, making them a promising pharmacological target against this debilitating disease. Molecular biology and cell electrophysiology have been used to look for new forms of treatment aiming to reduce aggressiveness and the disease progress.

Frontoxins, three-finger toxins from Micrurus frontalis venom, decrease miniature endplate potential amplitude at frog neuromuscular junction.

Neurotoxicity is a major symptom of envenomation caused by Brazilian coral snake Micrurus frontalis. Due to the small amount of material that can be collected, no neurotoxin has been fully sequenced from this venom. In this work we report six new three-finger like toxins isolated from the venom of the coral snake M. frontalis which we named Frontoxin (FTx) I-VI. Toxins were purified using multiple steps of RP-HPLC. Molecular masses were determined by MALDI-TOF and ESI ion-trap mass spectrometry. The complete amino acid sequence of FTx II, III, IV and V were determined by sequencing of overlapping proteolytic fragments by Edman degradation and by de novo sequencing. The amino acid sequences of FTx I, II, III and VI predict 4 conserved disulphide bonds and structural similarity to previously reported short-chain alpha-neurotoxins. FTx IV and V each contained 10 conserved cysteines and share high similarity with long-chain alpha-neurotoxins. At the frog neuromuscular junction FTx II, III and IV reduced miniature endplate potential amplitudes in a time-and concentration-dependent manner suggesting Frontoxins block nicotinic acetylcholine receptors.

A new family of small (4kDa) neurotoxins from the venoms of spiders of the genus Phoneutria.

A family of 4kDa neurotoxic peptides was purified from venoms of Phoneutria spiders. All have six cysteine residues, and low similarity with other neurotoxins. Three toxins caused moderate inhibition of L-type Ca(2+) channels. The structure of toxin PRTx27C3 was modeled and compared with toxin ADO1. The importance of four residues is suggested.

Comparison of the partial proteomes of the venoms of Brazilian spiders of the genus Phoneutria.

The proteomes of the venoms of the Brazilian wandering "armed" spiders Phoneutria nigriventer, Phoneutria reidyi, and Phoneutria keyserlingi, were compared using two-dimensional gel electrophoresis. The venom components were also fractionated using a combination of preparative reverse phase HPLC on Vydac C4, analytical RP-HPLC on Vydac C8 and C18 and cation exchange FPLC on Resource S at pH 6.1 and 4.7, or anion exchange HPLC on Synchropak AX-300 at pH 8.6. The amino acid sequences of the native and S-pyridyl-ethylated proteins and peptides derived from them by enzymatic digestion and chemical cleavages were determined using a Shimadzu PPSQ-21(A) automated protein sequencer, and by MS/MS collision induced dissociations. To date nearly 400 peptides and proteins (1.2-27 kDa) have been isolated in a pure state and, of these, more than 100 have had their complete or partial amino acid sequences determined. These sequences demonstrate, as might be expected, that the venoms of P. reidyi and P. keyserlingi (Family: Ctenidae) both contain a similar range of isoforms of the neurotoxins as those previously isolated from P. nigriventer which are active on neuronal ion (Ca(2+), Na(+) and K(+)) channels and NMDA-type glutamate receptors. In addition two new families of small (3-4 kDa) toxins, some larger protein (>10 kDa) components, and two serine proteinases of the venom of P. nigriventer are described. These enzymes may be responsible for some of the post-translational modification observed in some of the venom components.

CaV 3.1 and CaV 3.3 account for T-type Ca2+ current in GH3 cells.

T-type Ca2+ channels are important for cell signaling by a variety of cells. We report here the electrophysiological and molecular characteristics of the whole-cell Ca2+ current in GH3 clonal pituitary cells. The current inactivation at 0 mV was described by a single exponential function with a time constant of 18.32 +/- 1.87 ms (N = 16). The I-V relationship measured with Ca2+ as a charge carrier was shifted to the left when we applied a conditioning pre-pulse of up to -120 mV, indicating that a low voltage-activated current may be present in GH3 cells. Transient currents were first activated at -50 mV and peaked around -20 mV. The half-maximal voltage activation and the slope factors for the two conditions are -35.02 +/- 2.4 and 6.7 +/- 0.3 mV (pre-pulse of -120 mV, N = 15), and -27.0 +/- 0.97 and 7.5 +/- 0.7 mV (pre-pulse of -40 mV, N = 9). The 8-mV shift in the activation mid-point was statistically significant (P < 0.05). The tail currents decayed bi-exponentially suggesting two different T-type Ca2+ channel populations. RT-PCR revealed the presence of alpha1G (CaV3.1) and alpha1I (CaV3.3) T-type Ca2+ channel mRNA transcripts.

CaV 3.1 and CaV 3.3 account for T-type Ca2+ current in GH3 cells

T-type Ca2+ channels are important for cell signaling by a variety of cells. We report here the electrophysiological and molecular characteristics of the whole-cell Ca2+ current in GH3 clonal pituitary cells. The current inactivation at 0 mV was described by a single exponential function with a time constant of 18.32 ñ 1.87 ms (N = 16). The I-V relationship measured with Ca2+ as a charge carrier was shifted to the left when we applied a conditioning pre-pulse of up to -120 mV, indicating that a low voltage-activated current may be present in GH3 cells. Transient currents were first activated at -50 mV and peaked around -20 mV. The half-maximal voltage activation and the slope factors for the two conditions are -35.02 ñ 2.4 and 6.7 ñ 0.3 mV (pre-pulse of -120 mV, N = 15), and -27.0 ñ 0.97 and 7.5 ñ 0.7 mV (pre-pulse of -40 mV, N = 9). The 8-mV shift in the activation mid-point was statistically significant (P < 0.05). The tail currents decayed bi-exponentially suggesting two different T-type Ca2+ channel populations. RT-PCR revealed the presence of a1G (CaV3.1) and a1I (CaV3.3) T-type Ca2+ channel mRNA transcripts.

Electrophysiological evidence for glial-subtype glutamate transporter functional expression in rat cerebellar granule neurons.

A glutamate-sensitive inward current (Iglu) is described in rat cerebellar granule neurons and related to a glutamate transport mechanism. We examined the features of Iglu using the patch-clamp technique. In steady-state conditions the Iglu measured 8.14 1.9 pA. Iglu was identified as a voltage-dependent inward current showing a strong rectification at positive potentials. L-Glutamate activated the inward current in a dose-dependent manner, with a half-maximal effect at about 18 M and a maximum increase of 51.2 4.4%. The inward current was blocked by the presence of dihydrokainate (0.5 mM), shown by others to readily block the GLT1 isoform. We thus speculate that Iglu could be attributed to the presence of a native glutamate transporter in cerebellar granule neurons.

Electrophysiological evidence for glial-subtype glutamate transporter functional expression in rat cerebellar granule neurons

A glutamate-sensitive inward current (Iglu) is described in rat cerebellar granule neurons and related to a glutamate transport mechanism. We examined the features of Iglu using the patch-clamp technique. In steady-state conditions the Iglu measured 8.14 ± 1.9 pA. Iglu was identified as a voltage-dependent inward current showing a strong rectification at positive potentials. L-Glutamate activated the inward current in a dose-dependent manner, with a half-maximal effect at about 18 æM and a maximum increase of 51.2 ± 4.4 percent. The inward current was blocked by the presence of dihydrokainate (0.5 mM), shown by others to readily block the GLT1 isoform. We thus speculate that Iglu could be attributed to the presence of a native glutamate transporter in cerebellar granule neurons

Regulation of the glutamate uptake by extracellular calcium.

To determine whether [Ca(2+)](e) modulates glutamate re-uptake, we studied the uptake mechanism into rat cerebrocortical synaptosomes. The removal of extracellular Ca(2+) caused a negative modulation in the uptake mechanism. The calculated K(50) value was 0.185 +/- 0.019 mM (n = 4). The Michaelis-Menten data analysis indicate that absence of Ca(2+) diminished the V(max) kinetic parameter by about 60% without changing significantly the K(m) suggesting a non-competitive mechanism. We also tested the involvement of intracellular Ca(2+) in this phenomenon by trapping BAPTA into the synaptosomal vesicles to control the Ca(2+) concentration. Our results suggest that intracellular Ca(2+) changes have a less predominant role on the glutamate uptake than do extracellular Ca(2+). These findings argue in favor of an important role of extracellular [Ca(2+)] in maintaining the L-glutamate re-uptake mechanism in the mammalian central nervous system.

Partial purification and pharmacological characterization of a neurotoxic fraction isolet from the venom of the spider Lycosa erythrognatha

The effect of the venom of the spider Lycosa erythrognatha on the frog sciatic nerve was investigated with the single sucrose-gap method. Solutions containing the crude venom (40 µg protein/ml) markedly increased the duration of compound action potentials and caused the appearance of long-lasting depolarizing post-potentials. These effects were only partially (20 percent) reversded by extensive washsing with control solution. The active material was sensitive to proteolytic treatments with pronase or trypsin and was separated with 20 percent acetonitrile and 0.1 percent trifluoroacetic acid by reverse phase chromatography. The effect of this fraction (LycIV) on the post-potential amplitude was concentration-dependent, and was fitted with a quadratic hyperbola having a half maximal effect of 0.9 µg protein/ml. SDS-polyacrylamide gel electrophoresis of LycIV showed an enriched polypeptide band with apparent molecular weight of ~8 kDa. The observed effects were similar to those of toxins that inhibit sodium channel inactivation and different from the effects of potassium channel blockers. Pore formation or membrane disruption could be ruled out. It was concluded that the venom contains a neurotoxic polypeptide that alters the repolarization of action potentials, probably by inhibiting sodium channel inactivation

Effects of crotoxin on the action potential kinetics of frog skeletal muscle

1. The effect of crotoxin on the action potential kinetics of frog (Rana catesbeiana; 60-80 g) skeletal muscle was studied using a modified Ringer solution containing 1.6 mM KCl. 2. Crotoxin affected the kinetics of the action potential in a dose-dependent manner (90 to 460 nM). At 230 nM, crotoxin prolonged the duration of the action potential (from 1.1 +/- 0.1 to 1.6 +/- 0.1 ms) and slowed the rates of depolarization (from 282.0 +/- 7.3 to 196.0 +/- 13.2 V/s) and repolarization (from -81.4 +/- 6.9 to -55.6 +/- 3.8 V/s), in a dose-dependent manner. Its phospholipase subunit (component B) was five times less effective. 3. No effect of crotoxin was observed in the presence of 2.5 mM KCl or when SrCl2 was substituted for CaCl2. Lowering the muscle temperature to 12 degrees C did not reduce the effect of crotoxin. 4. No effect on the passive membrane response to hyperpolarizing current pulses was observed, which implies no major effect on the membrane resistance and capacitance. 5. It is concluded that crotoxin reduces the Na+ current and slows down the repolarization mechanism. This effect is probably not dependent on the phospholipase A2 activity of crotoxin and is inhibited by the substitution of Sr2+ for Ca2+

The effect of crotoxin on the release of acetylcholine and lactate dehydrogenase from rat brain cortical slices

We have studied the effects of crotoxin, the neurotoxin of the South American rattlesnake Crotalus durissus terrificus, ,on the release of acetylcholine and lactate dehydrogenase from rat brain cortical slices. Crotoxin enhances the release of [3H]-acetylcholine from cortical slices (control values 92.8 ñ 5.9 and 150.3 ñ 11.7 DPM/mg and crotoxin valuesw 199.1 ñ 7.0 and 336.0 ñ 26.0 DPM/mg at 60 and 120 min incubation, respectively) in parallel with the release of lactate dehydrogenase (control values 50.4 ñ 16.8 and 80.3 ñ 19.5 U/mg and crotoxin values 162.5 ñ 39.1 and 355.7 ñ 38.2 U/mg, at 120 min incubation, respectively). Both effects are markedly reduced when substituting Sr2+ for Ca2+ in the incubation medium. It is concluded that the phospholipase activity of crotoxin is responsible for the observed effects