Cardiac K+ channels are membrane-spanning proteins that allow the passive movement of K+ ions across the cell membrane along its electrochemical gradient. They regulate the resting membrane potential, the frequency of pacemaker cells and the shape and duration of the cardiac action potential. Additionally, they have been recognized as potential targets for the actions of neurotransmitters and hormones and class III antiarrhythmic drugs that prolong the action potential duration (APD) and refractoriness and have been found effective to prevent/suppress cardiac arrhythmias. In the human heart, K+ channels include voltage-gated channels, such as the rapidly activating and inactivating transient outward current (Ito1), the ultrarapid (IKur), rapid (IKr) and slow (IKs) components of the delayed rectifier current and the inward rectifier current (IK1), the ligand-gated channels, including the adenosine triphosphate-sensitive (IKATP) and the acetylcholine-activated (IKAch) currents and the leak channels. Changes in the expression of K+ channels explain the regional variations in the morphology and duration of the cardiac action potential among different cardiac regions and are influenced by heart rate, intracellular signalling pathways, drugs and cardiovascular disorders. A progressive number of cardiac and noncardiac drugs block cardiac K+ channels and can cause a marked prolongation of the action potential duration (i.e. an acquired long QT syndrome, LQTS) and a distinct polymorphic ventricular tachycardia termed torsades de pointes. In addition, mutations in the genes encoding IKr (KCNH2/KCNE2) and IKs (KCNQ1/KCNE1) channels have been identified in some types of the congenital long QT syndrome. This review concentrates on the function, molecular determinants, regulation and, particularly, on the mechanism of action of drugs modulating the K+ channels present in the sarcolemma of human cardiac myocytes that contribute to the different phases of the cardiac action potential under physiological and pathological conditions.

Stereoselective drug-channel interactions may help to elucidate the molecular basis of voltage-gated potassium channel block by local anesthetic drugs. We studied the effects of the enantiomers of bupivacaine on a cloned human cardiac potassium channel (hKv1.5). This channel was stably expressed in a mouse Ltk- cell line and studied using the whole-cell configuration of the patch-clamp technique. Both enantiomers modified the time course of this delayed rectifier current. Exposure to 20 microM of either S(-)-bupivacaine or R(+)-bupivacaine did not modify the activation time constant of the current, but reduced the peak outward current and induced a subsequent exponential decline of current with time constants of 18.7 +/- 1.1 and 10.0 +/- 0.9 ms, respectively. Steady-state levels of block (assessed with 250-ms depolarizing pulses to +60 mV) averaged 30.8 +/- 2.5% (n = 6) and 79.5 +/- 3.2% (n = 6) (p < 0.001), for S(-)- and R(+)-bupivacaine, respectively. The concentration dependence of hKv1.5 inhibition revealed apparent KD values of 27.3 +/- 2.8 and 4.1 +/- 0.7 microM for S(-)-bupivacaine and R(+)-bupivacaine, respectively, with Hill coefficients close to unity, suggesting that binding of one enantiomer molecule per channel was sufficient to block potassium permeation. Analysis of the rate constants of association (k) and dissociation (l) yielded similar values for l (24.9 s-1 vs. 23.6 s-1 for S(-)- and R(+)-bupivacaine, respectively) but different association rate constants (1.0 x 10(6) vs. 4.7 x 10(6) M-1 s-1 for S(-)- and R(+)-bupivacaine, respectively). Block induced by either enantiomer displayed a shallow voltage dependence in the voltage range positive to 0 mV, i.e., where the channel is fully open, consistent with an equivalent electrical distance delta of 0.16 +/- 0.01. This suggested that at the binding site, both enantiomers of bupivacaine experienced 16% of the applied transmembrane electrical field, referenced to the inner surface. Both bupivacaine enantiomers reduced the tail current amplitude recorded on return to -40 mV and slowed their time course relative to control, resulting in a "crossover" phenomenon. These data indicate 1) the charged form of both bupivacaine enantiomers block the hKv1.5 channel after it opens, 2) binding occurs within the transmembrane electrical field, 3) unbinding is required before the channel can close, 4) block of hKv1.5 channels by bupivacaine is markedly stereoselective, with the R(+)-enantiomer being the more potent one, 5) this stereoselective block was associated with a 1.11 -kcal/mol difference in binding energy between both enantiomers, and 6) the stereoselectivity derives mainly from a difference in the association rate constants, suggesting that the S(-)-enantiomer is less likely to access the binding site in an optimal configuration.

Both the large level of pulse-dependent block and the failure of the pure inactivated-state block model indicate that bupivacaine interacts with the activated (or open) state of the cardiac sodium channel in addition to its block of the inactivated state. The bupivacaine-induced block of the inactivated state of the Na+ channel displayed stereoselectivity, with R(+)-bupivacaine interacting faster and more potently. Both enantiomers also bind with high affinity to the activated or open state of the channel, but this interaction did not display stereoselectivity, although the binding to the activated or open state was faster for S(-)- than for R(+)-bupivacaine. The higher potency of R(+)-bupivacaine to block the inactivated state of the cardiac Na+ channel may explain its higher toxicity because of the large contribution of the inactivated-state block during the plateau phase of the cardiac action potential. These results would support the use of the S(-)-enantiomer to reduce cardiac toxicity.

Enantiomers of local anesthetics are useful probes of ion channel structure that can reveal three-dimensional relations for drug binding in the channel pore and may have important clinical consequences. Bupivacaine block of open hKv1.5 channels is stereoselective, with the R(+)-enantiomer being 7-fold more potent than the S(-)-enantiomer (Kd = 4.1 mumol/L versus 27.3 mumol/L). Using whole-cell voltage clamp of hKv1.5 channels and site-directed mutants stably expressed in Ltk- cells, we have identified a set of amino acids that determine the stereoselectivity of bupivacaine block. Replacement of threonine 505 by hydrophobic amino acids (isoleucine, valine, or alanine) abolished stereoselective block, whereas a serine substitution preserved it [Kd = 60 mumol/L and 7.4 mumol/L for S(-)- and R(+)-bupivacaine, respectively]. A similar substitution at the internal tetraethylammonium binding site (T477S) reduced the affinity for both enantiomers similarly, thus preserving the stereoselectivity [Kd = 45.5 mumol/L and 7.8 mumol/L for S(-)- and R(+)-bupivacaine, respectively]. Replacement of L508 or V512 by a methionine (L508M and V512M) abolished stereoselective block, whereas substitution of V512 by an alanine (V512A) preserved it. Block of Kv2.1 channels, which carry valine, leucine, and isoleucine residues at T505, L508, and V512 equivalent sites, respectively, was not stereoselective [Kd = 8.3 mumol/L and 13 mumol/L for S(-)- and R(+)-bupivacaine, respectively]. These results suggest that (1) the bupivacaine binding site is located in the inner mouth of the pore, (2) stereoselective block displays subfamily selectivity, and (3) a polar interaction with T505 combined with hydrophobic interactions with L508 and V512 are required for stereoselective block.

Deregulated protein and Ca2+ homeostasis underlie synaptic dysfunction and neurodegeneration in Huntington disease (HD); however, the factors that disrupt homeostasis are not fully understood. Here, we determined that expression of downstream regulatory element antagonist modulator (DREAM), a multifunctional Ca2+-binding protein, is reduced in murine in vivo and in vitro HD models and in HD patients. DREAM downregulation was observed early after birth and was associated with endogenous neuroprotection. In the R6/2 mouse HD model, induced DREAM haplodeficiency or blockade of DREAM activity by chronic administration of the drug repaglinide delayed onset of motor dysfunction, reduced striatal atrophy, and prolonged life span. DREAM-related neuroprotection was linked to an interaction between DREAM and the unfolded protein response (UPR) sensor activating transcription factor 6 (ATF6). Repaglinide blocked this interaction and enhanced ATF6 processing and nuclear accumulation of transcriptionally active ATF6, improving prosurvival UPR function in striatal neurons. Together, our results identify a role for DREAM silencing in the activation of ATF6 signaling, which promotes early neuroprotection in HD.

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.