Voltage-dependent potassium (Kv) channels play a pivotal role in the modulation of macrophage physiology. Macrophages are professional antigen-presenting cells and produce inflammatory and immunoactive substances that modulate the immune response. Blockage of Kv channels by specific antagonists decreases macrophage cytokine production and inhibits proliferation. Numerous pharmacological agents exert their effects on specific target cells by modifying the activity of their plasma membrane ion channels. Investigation of the mechanisms involved in the regulation of potassium ion conduction is, therefore, essential to the understanding of potassium channel functions in the immune response to infection and inflammation. Here, we demonstrate that the biophysical properties of voltage-dependent K+ currents are modified upon activation or immunosuppression in macrophages. This regulation is in accordance with changes in the molecular characteristics of the heterotetrameric Kv1.3/Kv1.5 channels, which generate the main Kv in macrophages. An increase in K+ current amplitude in lipopolysaccharide-activated macrophages is characterized by a faster C-type inactivation, a greater percentage of cumulative inactivation, and a more effective margatoxin (MgTx) inhibition than control cells. These biophysical parameters are related to an increase in Kv1.3 subunits in the Kv1.3/Kv1.5 hybrid channel. In contrast, dexamethasone decreased the C-type inactivation, the cumulative inactivation, and the sensitivity to MgTx concomitantly with a decrease in Kv1.3 expression. Neither of these treatments apparently altered the expression of Kv1.5. Our results demonstrate that the immunomodulation of macrophages triggers molecular and biophysical consequences in Kv1.3/Kv1.5 hybrid channels by altering the subunit stoichiometry.

This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. A c c e p t e d M a n u s c r i p t 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 and IL-2 production in stimulated Jurkat T-cells were also blocked by pharmacological doses of diclofenac. These effects were mimicked by Margatoxin, a specific Kv1.3 inhibitor, and Charybdotoxin, which blocks both Kv1.3 and Ca 2+ -activated K + channels (K Ca 3.1). Because Kv1.3 is a very good target for autoimmune therapies, the effects of diclofenac on Kv1.3 are of high pharmacological relevance.

Voltage-dependent K(+) channels (Kv) are involved in the proliferation of many types of cells, but the mechanisms by which their activity is related to cell growth remain unclear. Kv antagonists inhibit the proliferation of mammalian cells, which is of physiological relevance in skeletal muscle. Although myofibres are terminally differentiated, some resident myoblasts may re-enter the cell cycle and proliferate. Here we report that the expression of Kv1.5 is cell-cycle dependent during myoblast proliferation. In addition to Kv1.5 other Kv, such as Kv1.3, are also up-regulated. However, pharmacological evidence mainly implicates Kv1.5 in myoblast growth. Thus, the presence of S0100176, a Kv antagonist, but not margatoxin and dendrotoxin, led to cell cycle arrest during the G(1)-phase. The use of selective cell cycle blockers showed that Kv1.5 was transiently accumulated during the early G(1)-phase. Furthermore, while myoblasts treated with S0100176 expressed low levels of cyclin A and D(1), the expression of p21(cip-1) and p27(kip1), two cyclin-dependent kinase inhibitors, increased. Our results indicate that the cell cycle-dependent expression of Kv1.5 is involved in skeletal muscle cell proliferation.

Dietary polyunsaturated fatty acids (PUFAs) have been reported to exhibit antiarrhythmic properties, which have been attributed to their availability to modulate Na(+), Ca(2+), and several K(+) channels. However, their effects on human ether-a-go-go-related gene (HERG) channels are unknown. In this study we have analyzed the effects of arachidonic acid (AA, omega-6) and docosahexaenoic acid (DHA, omega-3) on HERG channels stably expressed in Chinese hamster ovary cells by using the whole cell patch-clamp technique. At 10 microM, AA and DHA blocked HERG channels, at the end of 5-s pulses to -10 mV, to a similar extent (37.7 +/- 2.4% vs. 50.2 +/- 8.1%, n = 7-10, P > 0.05). 5,6,11,14-Eicosatetrayenoic acid, a nonmetabolizable AA analog, induced effects similar to those of AA on HERG current. Both PUFAs shifted the midpoint of activation curves of HERG channels by -5.1 +/- 1.8 mV (n = 10, P < 0.05) and -11.2 +/- 1.1 mV (n = 7, P < 0.01). Also, AA and DHA shifted the midpoint of inactivation curves by +12.0 +/- 3.9 mV (n = 4; P < 0.05) and +15.8 +/- 4.3 mV (n = 4; P < 0.05), respectively. DHA and AA accelerated the deactivation kinetics and slowed the inactivation kinetics at potentials positive to +40 mV. Block induced by DHA, but not that produced by AA, was higher when measured after applying a pulse to -120 mV (I-->O). Finally, both AA and DHA induced a use-dependent inhibition of HERG channels. In summary, block induced by AA and DHA was time, voltage, and use dependent. The results obtained suggest that both PUFAs bind preferentially to the open state of the channel, although an interaction with inactivated HERG channels cannot be ruled out for AA.

Rationale:The mutation A341V in the S6 transmembrane segment of KCNQ1, the ␣-subunit of the slowly activating delayed-rectifier K ؉ (I Ks ) channel, predisposes to a severe long-QT1 syndrome with sympathetictriggered ventricular tachyarrhythmias and sudden cardiac death.Objective: Several genetic risk modifiers have been identified in A341V patients, but the molecular mechanisms underlying the pronounced repolarization phenotype, particularly during ␤-adrenergic receptor stimulation, remain unclear. We aimed to elucidate these mechanisms and provide new insights into control of cAMPdependent modulation of I Ks . Methods and Results:We characterized the effects of A341V on the I Ks macromolecular channel complex in transfected Chinese hamster ovary cells and found a dominant-negative suppression of cAMP-dependent Yotiao-mediated I Ks upregulation on top of a dominant-negative reduction in basal current. Phosphomimetic substitution of the N-terminal position S27 with aspartic acid rescued this loss of upregulation. Western blot analysis showed reduced phosphorylation of KCNQ1 at S27, even for heterozygous A341V, suggesting that phosphorylation defects in some (mutant) KCNQ1 subunits can completely suppress I Ks upregulation. Functional analyses of heterozygous KCNQ1 WT:G589D and heterozygous KCNQ1 WT:S27A, a phosphorylation-inert substitution, also showed such suppression. Immunoprecipitation of Yotiao with KCNQ1-A341V (in the presence of KCNE1) was not different from wild-type. Key Words: ion channels Ⅲ long-QT syndrome Ⅲ potassium Ⅲ torsade de pointes T he slowly activating delayed-rectifier K ϩ current (I Ks ) contributes importantly to cardiac repolarization. In large mammals, including humans, it has a small amplitude under basal isolated-myocyte conditions but forms a sizable repolarization reserve that is recruited when the action potential duration (APD) prolongs and during ␤-adrenergic receptor (␤AR) stimulation. 1,2 I Ks is carried by a macromolecular channel complex consisting of a homotetramer of pore-forming ␣-subunits encoded by KCNQ1 (Kv7.1), KCNE1 ␤-subunits, 3,4 and the regulatory A-kinase anchoring protein Yotiao, which binds to the KCNQ1 C-terminus. 5 There are also multiple interactions with other proteins. During ␤AR stimulation, when cAMP levels rise, phosphorylation of KCNQ1 at N-terminal position S27 is controlled by protein kinase A (PKA) and protein phosphatase 1 that are localized to the complex by Yotiao, thereby providing local control of I Ks enhancement. 5 Anchored PKA also phosphorylates Yotiao itself, thereby further enhancing I Ks . 6 An intact C-terminus of KCNE1 is critical for the PKA-dependent upregulation of I Ks . 7 Congenital defects (long-QT syndrome types 1 and 5; LQT1, LQT5; Jervell and Lange-Nielsen syndrome), pharmacological inhibition, 8 and acquired loss of I Ks , 9 can all result in QT-interval prolongation and enhanced susceptibility to ventricular tachyarrhythmias, notably torsade de pointes. These arrhythmias occur predominantly during conditions of exerc...

The degree of stereoselective block of Kv1.5 decreases from 9 to 4 when Kvbeta1.3 is present. L510 is determinant for the modulation of bupivacaine block, because it is the only residue of the S6 segment that binds to both bupivacaine and Kvbeta1.3. These findings support an overlapping binding site for drugs and Kvbeta1.3.

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Irvalec is a marine-derived antitumor agent currently undergoing phase II clinical trials. In vitro, Irvalec induces a rapid loss of membrane integrity in tumor cells, accompanied of a significant Ca2+ influx, perturbations of membrane conductivity, severe swelling and the formation of giant membranous vesicles. All these effects are not observed in Irvalec-resistant cells, or are significantly delayed by pretreating the cells with Zn2+. Using fluorescent derivatives of Irvalec it was demonstrated that the compound rapidly interacts with the plasma membrane of tumor cells promoting lipid bilayer restructuration. Also, FRET experiments demonstrated that Irvalec molecules localize in the cell membrane close enough to each other as to suggest that the compound could self-organize, forming supramolecular structures that likely trigger cell death by necrosis through the disruption of membrane integrity.