Although chloroquine remains an important therapeutic agent for treatment of malaria in many parts of the world, its safety margin is very narrow. Chloroquine inhibits the cardiac inward rectifier K ؉ current IK1 and can induce lethal ventricular arrhythmias. In this study, we characterized the biophysical and molecular basis of chloroquine block of Kir2.1 channels that underlie cardiac I K1. The voltage-and K ؉ -dependence of chloroquine block implied that the binding site was located within the ion-conduction pathway. Site-directed mutagenesis revealed the location of the chloroquine-binding site within the cytoplasmic pore domain rather than within the transmembrane pore. Molecular modeling suggested that chloroquine blocks Kir2.1 channels by plugging the cytoplasmic conduction pathway, stabilized by negatively charged and aromatic amino acids within a central pocket. Unlike most ionchannel blockers, chloroquine does not bind within the transmembrane pore and thus can reach its binding site, even while polyamines remain deeper within the channel vestibule. These findings explain how a relatively low-affinity blocker like chloroquine can effectively block I K1 even in the presence of high-affinity endogenous blockers. Moreover, our findings provide the structural framework for the design of safer, alternative compounds that are devoid of Kir2.1-blocking properties.IK1 ͉ ion channel ͉ KCNJ2 ͉ malaria ͉ polyamines

Voltage-gated K + (Kv) channels couple the movement of a voltage sensor to the channel gate(s) via a helical intracellular region, the S4-S5 linker. A number of studies link voltage sensitivity to interactions of S4 charges with membrane phospholipids in the outer leaflet of the bilayer. Although the phospholipid phosphatidylinositol-4,5-bisphosphate (PIP 2 ) in the inner membrane leaflet has emerged as a universal activator of ion channels, no such role has been established for mammalian Kv channels. Here we show that PIP 2 depletion induced two kinetically distinct effects on Kv channels: an increase in voltage sensitivity and a concomitant decrease in current amplitude. These effects are reversible, exhibiting distinct molecular determinants and sensitivities to PIP 2 . Gating current measurements revealed that PIP 2 constrains the movement of the sensor through interactions with the S4-S5 linker. Thus, PIP 2 controls both the movement of the voltage sensor and the stability of the open pore through interactions with the linker that connects them.voltage-gated channels | lipids | channel modulation | open probability V oltage-gated K + (Kv) channels are tetrameric integral membrane proteins critical to membrane excitability that respond rapidly to changes in membrane potential to control membrane permeability to potassium ions. Upon membrane depolarization, a voltage sensor in each subunit undergoes a transition from a resting to an activated state followed by a concerted transition leading to the opening of the pore (1-4). The voltage-sensing domain [i.e., the S1-S4 transmembrane (TM) helices] of Kv channel subunits harbors within its S4 helix several positively charged residues that respond directly to changes in membrane voltage (5-7). The movement of these charges can be monitored by the gating current they produce, and the opening of the pore is monitored by the ionic current that follows. The S4-S5 linker couples the movement of the voltage sensor to the opening of the pore.X-ray structures of Kv channels have shown that the S1-S4 voltage-sensing domains are exposed to lipids when embedded in a membrane (8,9). A number of studies have suggested that, after depolarization, interactions of the S4 charges with lipids in the outer leaflet of the membrane are important in the stabilization of the sensor in the activated state (10-12).Phosphatidylinositol-4,5-bisphosphate (PIP 2 ), a phospholipid that affects the activity of many types of ion channels (13, 14), acts as a docking platform for the N-terminal domain of fastinactivating Kv channels (15). Activation of Ciona intestinalis voltage-sensitive phosphatase (Ci-VSP), which contains a voltage-sensing domain (S1-S4) coupled to a cytoplasmic phosphatase domain rather than a TM pore, shows a dependence on membrane depolarization similar to that of voltage-gated channels (16). PIP 2 modulates the motions of the Ci-VSP voltagesensor domain and its coupling to the phosphatase domain by interacting with the linker that connects the voltage sensor and phosphatase...

Tamoxifen, an estrogen receptor antagonist used in the treatment of breast cancer, inhibits the inward rectifier potassium current (I K1 ) in cardiac myocytes by an unknown mechanism. We characterized the inhibitory effects of tamoxifen on Kir2.1, Kir2.2, and Kir2.3 potassium channels that underlie cardiac I K1 . We also studied the effects of 4-hydroxytamoxifen and raloxifene. All three drugs inhibited inward rectifier K ϩ 2.x (Kir2.x) family members. The order of inhibition for all three drugs was Kir2.3 Ͼ Kir2.1 ϳ Kir2.2. The onset of inhibition of Kir2.x current by these compounds was slow (T 1/2 ϳ 6 min) and only partially recovered after washout (ϳ30%). Kir2.x inhibition was concentration-dependent but voltage-independent. The time course and degree of inhibition was independent of external or internal drug application. We tested the hypothesis that tamoxifen interferes with the interaction between the channel and the membrane-delimited channel activator, phosphatidylinositol 4,5-bisphosphate (PIP 2 ). Inhibition of Kir2.3 currents was significantly reduced by a single point mutation of I213L, which enhances Kir2.3 interaction with membrane PIP 2 . Pretreatment with PIP 2 significantly decreased the inhibition induced by tamoxifen, 4-hydroxytamoxifen, and raloxifene on Kir2.3 channels. Pretreatment with spermine (100 M) decreased the inhibitory effect of tamoxifen on Kir2.1, probably by strengthening the channel's interaction with PIP 2 . In cat atrial and ventricular myocytes, 3 M tamoxifen inhibited I K1 , but the effect was greater in the former than the latter. The data strongly suggest that tamoxifen, its metabolite, and the estrogen receptor inhibitor raloxifene inhibit Kir2.x channels indirectly by interfering with the interaction between the channel and PIP 2 .

BACKGROUND AND PURPOSE Wherever they are located, dopamine transporters (DATs) clear dopamine (DA) from the extracellular milieu to help regulate dopaminergic signalling. Exposure to amphetamine (AMPH) increases extracellular DA in the synaptic cleft, which has been ascribed to DAT reverse transport. Increased extracellular DA prolongs postsynaptic activity and reinforces abuse and hedonic behaviour. EXPERIMENTAL APPROACH Xenopus laevis oocytes expressing human (h) DAT were voltage‐clamped and exposed to DA, R(‐)AMPH, or S(+)AMPH. KEY RESULTS At ‐60mV, near neuronal resting potentials, S(+)AMPH induced a depolarizing current through hDAT, which after removing the drug, persisted for more than 30 min. This persistent leak in the absence of S(+)AMPH was in contrast to the currents induced by R(‐)AMPH and DA, which returned to baseline immediately after their removal. Our data suggest that S(+)AMPH and Na+ carry the initial S(+)AMPH‐induced current, whereas Na+ and Cl‐ carry the persistent leak current. We propose that the persistent current results from the internal action of S(+)AMPH on hDAT because the temporal effect was consistent with S(+)AMPH influx, and intracellular S(+)AMPH activated the effect. The persistent current was dependent on Na+ and was blocked by cocaine. Intracellular injection of S(+)AMPH also activated a DA‐induced persistent leak current. CONCLUSIONS AND IMPLICATIONS We report a hitherto unknown action of S(+)AMPH on hDAT that potentially affects AMPH‐induced DA release. We propose that internal S(+)AMPH acts as a molecular stent that holds the transporter open even after external S(+)AMPH is removed. Amphetamine‐induced persistent leak currents are likely to influence dopaminergic signalling, DA release mechanisms, and amphetamine abuse.

Inwardly rectifying potassium (Kir) channels are gated by the membrane phospholipid phosphatidylinositol-4,5-bisphosphate (PIP2). Among them, Kir3 channel gating requires additional molecules, such as the βγ subunits of G proteins or intracellular sodium. Using an interactive computational-experimental approach, we show that sodium sensitivity of Kir channels involves the side-chains of an aspartate and a histidine located across from each other in a critical loop in the cytosolic domain, as well as the backbone carbonyls of two additional residues and a water molecule. The location of the coordination site in the vicinity of a conserved arginine shown to affect channel-PIP2 interactions suggests that sodium triggers a structural switch that frees the critical arginine. Mutations of the aspartate and the histidine that affect sodium sensitivity also enhance the channel’s sensitivity to PIP2. Furthermore, based on the molecular characteristics of the coordination site, we identify and confirm experimentally a novel sodium-sensitive phenotype in Kir5.1.