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Voltage-dependent potassium ([K.sub.v]) 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 [K.sub.v] 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.sup.+] currents are modified upon activation or immunosuppression in macrophages. This regulation is in accordance with changes in the molecular characteristics of the heterotetrameric [K.sub.v] 1.3/[K.sub.v] 1.5 channels, which generate the main [K.sub.v] in macrophages. An increase in [K.sup.+] 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 [K.sub.v] 1.3 subunits in the [K.sub.v] 1.3/[K.sub.v] 1.5 hybrid channel. In contrast, dexamethasone decreased the C-type inactivation, the cumulative inactivation, and the sensitivity to MgTx concomitantly with a decrease in [K.sub.v] 1.3 expression. Neither of these treatments apparently altered the expression of [K.sub.v] 1.5. Our results demonstrate that the immunomodulation of macrophages triggers molecular and biophysical consequences in [K.sub.v] 1.3/[K.sub.v] 1.5 hybrid channels by altering the subunit stoichiometry. doi/ 10.1085/jgp.200910334

Dietary polyunsaturated fatty acids (PUFAs) have been reported to exhibit antiarrhythmic properties, which have been attributed to their availability to modulate [Na.sup.+], Ca[2.sup.+], and several [K.sup.+] 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 [micro]M, AA and DHA blocked HERG channels, at the end of 5-s pulses to -10 mV, to a similar extent (37.7 [+ or -] 2.4% vs. 50.2 [+ or -] 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 [+ or -] 1.8 mV (n = 10, P < 0.05) and -11.2 [+ or -] 1.1 mV (n = 7, P < 0.01). Also, AA and DHA shifted the midpoint of inactivation curves by +12.0 [+ or -] 3.9 mV (n = 4; P < 0.05) and +15.8 [+ or -] 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 [right arrow] 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. [K.sup.+] channel; membrane currents; ion channels; arrhythmia; antiarrhythmics

Neutral sphingomyelinase (nSMase)-derived ceramide has been proposed as a mediator of hypoxic pulmonary vasoconstriction (HPV), a specific response of the pulmonary circulation. Voltage-gated K+ (Kv) channels are modulated by numerous vasoactive factors, including hypoxia, and their inhibition has been involved in HPV. Herein, we have analyzed the effects of ceramide on Kv currents and contractility in rat pulmonary arteries (PA) and in mesenteric arteries (MA). The ceramide analog C6-ceramide inhibited Kv currents in PA smooth muscle cells (PASMC). Similar effects were obtained after the addition of bacterial sphingomyelinase (SMase), indicating a role for endogenous ceramide in Kv channel regulation. Kv current was reduced by stromatoxin and diphenylphosphine oxide-1 (DPO-1), selective inhibitors of Kv2.1 and Kv1.5 channels, respectively. The inhibitory effect of ceramide was still present in the presence of stromatoxin or DPO-1, suggesting that this sphingolipid inhibited both components of the native Kv current. Accordingly, ceramide inhibited Kv1.5 and Kv2.1 channels expressed in Ltk-cells. Ceramide-induced effects were reduced in human embryonic kidney 293 cells expressing Kv1.5 channels but not the regulatory subunit Kvβ2.1. The nSMase inhibitor GW4869 reduced the thromboxane-endoperoxide receptor agonist U46619-induced, but not endothelin-1-induced pulmonary vasoconstriction that was partly restored after addition of exogenous ceramide. The PKC-ζ pseudosubstrate inhibitor (PKCζ-PI) inhibited the Kv inhibitory and contractile effects of ceramide. In MA ceramide had no effect on Kv currents and GW4869 did not affect U46619-induced contraction. The effects of SMase were also observed in human PA. These results suggest that ceramide represents a crucial signaling mediator in the pulmonary vasculature. [ABSTRACT FROM AUTHOR]

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 Kv 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. [ABSTRACT FROM AUTHOR]

Abstract: Objective: Cellular cardiomyoplasty using skeletal myoblasts is a promising therapy for myocardial infarct repair. Once transplanted, myoblasts grow, differentiate and adapt their electrophysiological properties towards more cardiac-like phenotypes. Voltage-dependent Na+ channels (Nav) are the main proteins involved in the propagation of the cardiac action potential, and their phenotype affects cardiac performance. Therefore, we examined the expression of Nav during proliferation and differentiation in skeletal myocytes. Methods and results: We used the rat neonatal skeletal myocyte cell line L6E9. Proliferation of L6E9 cells induced Nav1.4 and Nav1.5, although neither protein has an apparent role in cell growth. During myogenesis, Nav1.5 was largely induced. Electrophysiological and pharmacological properties, as well as mRNA expression, indicate that cardiac-type Nav1.5 accounts for almost 90% of the Na+ current in myotubes. Unlike in proliferation, this protein plays a pivotal role in myogenesis. The adoption of a cardiac-like phenotype is further supported by the increase in Nav1.5 colocalization in caveolae. Finally, we demonstrate that the treatment of myoblasts with neuregulin further increased Nav1.5 in skeletal myocytes. Conclusion: Our results indicate that skeletal myotubes adopt a cardiac-like phenotype in cell culture conditions and that the expression of Nav1.5 acts as an underlying molecular mechanism. [Copyright &y& Elsevier]

Rationale: The mutation A341V in the S6 transmembrane segment of KCNQ1, the α-subunit of the slowly activating delayed-rectifier K(+) (I(Ks)) channel, predisposes to a severe long-QT1 syndrome with sympathetic-triggered 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 β-adrenergic receptor stimulation, remain unclear. We aimed to elucidate these mechanisms and provide new insights into control of cAMP-dependent 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., Conclusions: Our results indicate the involvement of the KCNQ1-S6 region at/or around A341 in cAMP-dependent stimulation of I(Ks), a process that is under strong dominant-negative control, suggesting that tetrameric KCNQ1 phosphorylation is required. Specific long-QT1 mutations, including heterozygous A341V, disable this regulation.

Kv1.5 channels are homotetramers of alpha-pore subunits mainly present in human atrium and pulmonary vasculature. Thus, Kv1.5 is a pharmacological target for cardiovascular diseases. Kv beta 1.3 assemblies with Kv alpha 1.5 and modifies its gating and pharmacology. A further knowledge of alpha-beta interactions and pharmacology will lead a better design of new drugs.