The voltage-gated potassium channel Kv1.3 plays an apparent dual physiological role by participating in activation and proliferation of leukocytes as well as promoting apoptosis in several types of tumor cells. Therefore, Kv1.3 is considered a potential pharmacological target for immunodeficiency and cancer. Different cellular locations of Kv1.3, at the plasma membrane or the mitochondria, could be responsible for such duality. While plasma membrane Kv1.3 facilitates proliferation, the mitochondrial channel modulates apoptotic signaling. Several molecular determinants of Kv1.3 drive the channel to the cell surface, but no information is available about its mitochondrial targeting. Caveolins, which are able to modulate cell survival, participate in the plasma membrane targeting of Kv1.3. The channel, via a caveolin-binding domain (CBD), associates with caveolin 1 (Cav1), which localizes Kv1.3 to lipid raft membrane microdomains. The aim of our study was to understand the role of such interactions not only for channel targeting but also for cell survival in mammalian cells. By using a caveolin association-deficient channel (Kv1.3 CBD_less), we demonstrate here that while the Kv1.3–Cav1 interaction is responsible for the channel localization in the plasma membrane, a lack of such interaction accumulates Kv1.3 in the mitochondria. Kv1.3 CBD_less severely affects mitochondrial physiology and cell survival, indicating that a functional link of Kv1.3 with Cav1 within the mitochondria modulates the pro-apoptotic effects of the channel. Therefore, the balance exerted by these two complementary mechanisms fine-tune the physiological role of Kv1.3 during cell survival or apoptosis. Our data highlight an unexpected role for the mitochondrial caveolin–Kv1.3 axis during cell survival and apoptosis.

J. Neurochem.(2011)117, 1009–1019. Lidocaine is a local anaesthetic that blocks sodium channels, but also inhibits several ligand‐gated ion‐channels. The aim of this work was to unravel the mechanisms by which lidocaine blocksTorpedonicotinic receptors transplanted toXenopusoocytes. Acetylcholine‐elicited currents were reversibly blocked by lidocaine, in a concentration dependent manner. At doses lower than theIC₅₀, lidocaine blocked nicotinic receptors only at negative potentials, indicating an open‐channel blockade; the binding site within the channel was at about 30% of the way through the electrical field across the membrane. In the presence of higher lidocaine doses, nicotinic receptors were blocked both at positive and negative potentials, acetylcholine dose–response curve shifted to the right and lidocaine pre‐application, before its co‐application with acetylcholine, enhanced the current inhibition, indicating all together that lidocaine also blocked resting receptors; besides, it increased the current decay rate. When lidocaine, at low doses, was co‐applied with 2‐(triethylammonio)‐N‐(2,6‐dimethylphenyl) acetamide bromide, edrophonium or 1,5‐bis(4‐allyldimethylammoniumphenyl)pentan‐3‐one dibromide, which are quaternary‐ammonium molecules that also blocked nicotinic receptors, there was an additive inhibitory effect, indicating that these molecules bound to different sites within the channel pore. These results prove that lidocaine blocks nicotinic receptors by several independent mechanisms and evidence the diverse and complex modulation of this receptor by structurally related molecules.

The use of Xenopus oocytes in electrophysiological and biophysical research constitutes a long and successful story, providing major advances to the knowledge of the function and modulation of membrane proteins, mostly receptors, ion channels, and transporters. Earlier reports showed that these cells are capable of correctly expressing heterologous proteins after injecting the corresponding mRNA or cDNA. More recently, the Xenopus oocyte has become an outstanding host–cell model to carry out detailed studies on the function of fully-processed foreign membrane proteins after their microtransplantation to the oocyte. This review focused on the latter overall process of transplanting foreign membrane proteins to the oocyte after injecting plasma membranes or purified and reconstituted proteins. This experimental approach allows for the study of both the function of mature proteins, with their native stoichiometry and post-translational modifications, and their putative modulation by surrounding lipids, mostly when the protein is purified and reconstituted in lipid matrices of defined composition. Remarkably, this methodology enables functional microtransplantation to the oocyte of membrane receptors, ion channels, and transporters from different sources including human post-mortem tissue banks. Despite the large progress achieved over the last decades on the structure, function, and modulation of neuroreceptors and ion channels in healthy and pathological tissues, many unanswered questions remain and, most likely, Xenopus oocytes will continue to help provide valuable responses.

Fritillaria bulbs are used in Traditional Chinese Medicine to treat several illnesses. Peimine (Pm), an anti-inflammatory compound from Fritillaria, is known to inhibit some voltage-dependent ion channels and muscarinic receptors, but its interaction with ligand-gated ion channels remains unexplored. We have studied if Pm affects nicotinic acetylcholine receptors (nAChRs), since they play broad functional roles, both in the nervous system and non-neuronal tissues. Muscle-type nAChRs were incorporated to Xenopus oocytes and the action of Pm on the membrane currents elicited by ACh (I_AChs) was assessed. Functional studies were combined with virtual docking and molecular dynamics assays. Co-application of ACh and Pm reversibly blocked I_ACh, with an IC₅₀ in the low micromolar range. Pm inhibited nAChR by: (i) open-channel blockade, evidenced by the voltage-dependent inhibition of I_Ach, (ii) enhancement of nAChR desensitization, revealed by both an accelerated I_ACh decay and a decelerated I_ACh deactivation, and (iii) resting-nAChR blockade, deduced from the I_ACh inhibition elicited by Pm when applied before ACh superfusion. In good concordance, virtual docking and molecular dynamics assays demonstrated that Pm binds to different sites at the nAChR, mostly at the transmembrane domain. Thus, Pm from Fritillaria bulbs, considered therapeutic herbs, targets nAChRs with high affinity, which might account for its anti-inflammatory actions.

Nicotinic acetylcholine (ACh) receptors (nAChRs) are included among the targets of a variety of local anesthetics, although the molecular mechanisms of blockade are still poorly understood. Some local anesthetics, such as lidocaine, act on nAChRs by different means through their ability to present as both charged and uncharged molecules. Thus, we explored the mechanisms of nAChR blockade by tetracaine, which at physiological pH is almost exclusively present as a positively charged local anesthetic. The nAChRs from Torpedo electroplaques were transplanted to Xenopus oocytes and the currents elicited by ACh (I_AChs), either alone or co-applied with tetracaine, were recorded. Tetracaine reversibly blocked I_ACh, with an IC₅₀ (i.e., the concentration required to inhibit half the maximum I_ACh) in the submicromolar range. Notably, at very low concentrations (0.1 μM), tetracaine reduced I_ACh in a voltage-dependent manner, the more negative potentials produced greater inhibition, indicating open-channel blockade. When the tetracaine concentration was increased to 0.7 μM or above, voltage-independent inhibition was also observed, indicating closed-channel blockade. The I_ACh inhibition by pre-application of just 0.7 μM tetracaine before superfusion of ACh also corroborated the notion of tetracaine blockade of resting nAChRs. Furthermore, tetracaine markedly increased nAChR desensitization, mainly at concentrations equal or higher than 0.5 μM. Interestingly, tetracaine did not modify desensitization when its binding within the channel pore was prevented by holding the membrane at positive potentials. Tetracaine-nAChR interactions were assessed by virtual docking assays, using nAChR models in the closed and open states. These assays revealed that tetracaine binds at different sites of the nAChR located at the extracellular and transmembrane domains, in both open and closed conformations. Extracellular binding sites seem to be associated with closed-channel blockade; whereas two sites within the pore, with different affinities for tetracaine, contribute to open-channel blockade and the enhancement of desensitization, respectively. These results demonstrate a concentration-dependent heterogeneity of tetracaine actions on nAChRs, and contribute to a better understanding of the complex modulation of muscle-type nAChRs by local anesthetics. Furthermore, the combination of functional and virtual assays to decipher nAChR-tetracaine interactions has allowed us to tentatively assign the main nAChR residues involved in these modulating actions.