Recent molecular-dynamics simulations have suggested that the arginine-rich HIV Tat peptides translocate by destabilizing and inducing transient pores in phospholipid bilayers. In this pathway for peptide translocation, Arg residues play a fundamental role not only in the binding of the peptide to the surface of the membrane, but also in the destabilization and nucleation of transient pores across the bilayer. Here we present a molecular-dynamics simulation of a peptide composed of nine Args (Arg-9) that shows that this peptide follows the same translocation pathway previously found for the Tat peptide. We test experimentally the hypothesis that transient pores open by measuring ionic currents across phospholipid bilayers and cell membranes through the pores induced by Arg-9 peptides. We find that Arg-9 peptides, in the presence of an electrostatic potential gradient, induce ionic currents across planar phospholipid bilayers, as well as in cultured osteosarcoma cells and human smooth muscle cells. Our results suggest that the mechanism of action of Arg-9 peptides involves the creation of transient pores in lipid bilayers and cell membranes.

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Cellular energetic deregulation is widely known to produce an overproduction of acidic species in cancer cells. This acid overload must be counterbalanced with a high rate of H extrusion to maintain cell viability. In this sense, many H transporters have been reported to be crucial for cell survival and proposed as antineoplastic target. By the way, voltage-gated proton channels (Hv1) mediate highly selective H outward currents, capable to compensate acid burden in brief periods of time. This structure is canonically described acting as NADPH oxidase counterbalance in reactive oxygen species production. In this work, we show, for the first time in a oncohematologic cell line, that inhibition of Hv1 channels by Zn and the more selective blocker 2-(6-chloro-1H-benzimidazol-2-yl)guanidine (ClGBI) progressively decreases intracellular pH in resting conditions. This acidification is evident minutes after blockade and progresses under prolonged exposure (2, 17, and 48 h), and we firstly demonstrate that this is followed by cell death through apoptosis (annexin V binding). Altogether, these results contribute strong evidence that this channel might be a new therapeutic target in cancer.

Arachidonic acid (AA) is a polyunsaturated fatty acid involved in a complex network of cell signaling. It is well known that this fatty acid can directly modulate several cellular target structures, among them, ion channels. We explored the effects of AA on high conductance Ca(2+)- and voltage-dependent K(+) channel (BKCa) in vascular smooth muscle cells (VSMCs) where the presence of β1-subunit was functionally demonstrated by lithocholic acid activation. Using patch-clamp technique, we show at the single channel level that 10 μM AA increases the open probability (Po) of BKCa channels tenfold, mainly by a reduction of closed dwell times. AA also induces a left-shift in Po versus voltage curves without modifying their steepness. Furthermore, AA accelerates the kinetics of the voltage channel activation by a fourfold reduction in latencies to first channel opening. When AA was tested on BKCa channel expressed in HEK cells with or without the β1-subunit, activation only occurs in presence of the modulatory subunit. These results contribute to highlight the molecular mechanism of AA-dependent BKCa activation. We conclude that AA itself selectively activates the β1-associated BKCa channel, destabilizing its closed state probably by interacting with the β1-subunit, without modifying the channel voltage sensitivity. Since BKCa channels physiologically contribute to regulation of VSMCs contractility and blood pressure, we used the whole-cell configuration to show that AA is able to activate these channels, inducing significant cell hyperpolarization that can lead to VSMCs relaxation.

Through their control of cell membrane potential, potassium (K þ ) channels are among the best known regulators of vascular tone. This article discusses the expression and function of K þ channels in human umbilical artery smooth muscle cells (HUASMCs). We review the bibliographic reports and also present single-channel data recorded in freshly isolated cells. Electrophysiological properties of big conductance, voltage-and Ca 2þ -sensitive K þ channel and voltage-dependent K þ channels are clearly established in this vessel, where they are involved in contractile state regulation. Their role in the maintenance of membrane potential is an important control mechanism in the determination of the vessel diameter. Additionally, small conductance Ca 2þ -sensitive K þ channels, 2-pore domains K þ channels and inward rectifier K þ channels also appear to be present in HUASMCs, while intermediate conductance Ca 2þ -sensitive K þ channels and ATP-sensitive K þ channels could not be identified. In both cases, additional investigation is necessary to reach conclusive evidence of their expression and/or functional role in HUASMCs. Finally, we discuss the role of K þ channels in pregnancy-related pathologies like gestational diabetes and preeclampsia.

The identities of K+ channels in the human umbilical artery were shown using the patch-clamp technique, and the physiologic effect of K+ channels on resting tone was documented.

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The electrophysiological and pharmacological properties of Ca²⁺ current (I_Ca) were determined by the whole-cell configuration of the patch-clamp technique in smooth muscle cells from human umbilical artery. Using 5 mM extracellular Ca²⁺, depolarizing step pulses from -60 to 50 mV from a holding membrane potential of -80 mV evoked an I_Ca which activated at membrane potentials more positive than -50 mV and exhibited a maximum current density in a range of 10-20 mV. Steady-state inactivation protocols using a V_test of 10 mV gave a voltage at one-half inactivation and a slope factor of -35.6 mV and 9.5 mV, respectively. Nifedipine (1 µM), an L-type Ca²⁺ channels antagonist, completely inhibited I_Ca, while the L-type Ca²⁺ channels agonist Bay-K 8644 (1 µM) significantly increased I_Ca amplitude. Moreover, the selective blocker of P-/Q-type Ca²⁺ channels ω-agatoxin IVA partially blocked I_Ca (about 40 % inhibition at +20 mV by 20 nM). These pharmacological results suggest that L- and P-/Q-type Ca²⁺ channels, both nifedipine-sensitive, underlie the I_Ca registered using low extracellular Ca²⁺. The presence of the P-/Q-type Ca²⁺ channels was confirmed by immunoblot analysis. When I_Ca was recorded in a high concentration (30 mM) of extracellular Ca²⁺ or Ba²⁺ as current carrier, it was evident the presence of a nifedipine-insensitive component which completely inactivated during the course of the voltage-step (75 ms) at all potentials tested, and was blocked by the T-type Ca²⁺ channels blocker mibefradil (10 µM). Summarizing, this work shows for the first time the electrophysiological and pharmacological properties of voltage-activated Ca²⁺ currents in human umbilical artery smooth muscle cells.