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⁺currentI_K1and can induce lethal ventricular arrhythmias. In this study, we characterized the biophysical and molecular basis of chloroquine block of Kir2.1 channels that underlie cardiacI_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 ion-channel 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 blockI_K1even 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.

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.

Over the past 16 years, there has been an impressive number of ion channels shown to be sensitive to the major phosphoinositide in the plasma membrane, phosphatidylinositol 4,5-bisphosphate (PIP₂). Among them are voltage-gated channels, which are crucial for both neuronal and cardiac excitability. Voltage-gated calcium (Cav) channels were shown to be regulated bidirectionally by PIP₂. On one hand, PIP₂ stabilized their activity by reducing current rundown but on the other hand it produced a voltage-dependent inhibition by shifting the activation curve to more positive voltages. For voltage-gated potassium (Kv) channels PIP₂ was first shown to prevent N-type inactivation regardless of whether the fast inactivation gate was part of the pore-forming α subunit or of an accessory β subunit. Careful examination of the effects of PIP₂ on the activation mechanism of Kv1.2 has shown a similar bidirectional regulation as in the Cav channels. The two effects could be distinguished kinetically, in terms of their sensitivities to PIP₂ and by distinct molecular determinants. The rightward shift of the Kv1.2 voltage dependence implicated basic residues in the S4–S5 linker and was consistent with stabilization of the inactive state of the voltage sensor. A third type of a voltage-gated ion channel modulated by PIP₂ is the hyperpolarization-activated cyclic nucleotide-gated (HCN) channel. PIP₂ has been shown to enhance the opening of HCN channels by shifting their voltage-dependent activation toward depolarized potentials. The sea urchin HCN channel, SpIH, showed again a PIP₂-mediated bidirectional effect but in reverse order than the depolarization-activated Cav and Kv channels: a voltage-dependent potentiation, like the mammalian HCN channels, but also an inhibition of the cGMP-induced current activation. Just like the Kv1.2 channels, distinct molecular determinants underlied the PIP₂ dual effects on SpIH, with the proximal C-terminus implicated in the inhibitory effect. The dual regulation of these very different ion channels, all of which are voltage-dependent, points to conserved mechanisms of regulation of these channels by PIP₂.

Inwardly rectifying potassium (Kir) channels are gated by the membrane phospholipid phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P₂). Among them, Kir3 requires additional molecules, such as the βγ subunits of G proteins or intracellular sodium, for channel gating. 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 crucial loop in the cytosolic domain, as well as the backbone carbonyls of two more residues and a water molecule. The location of the coordination site in the vicinity of a conserved arginine shown to affect channel–PtdIns(4,5)P₂ interactions suggests that sodium triggers a structural switch that frees the crucial arginine. Mutations of the aspartate and the histidine that affect sodium sensitivity also enhance the channel's sensitivity to PtdIns(4,5)P₂. Furthermore, on the basis of the molecular characteristics of the coordination site, we identify and confirm experimentally a sodium-sensitive phenotype in Kir5.1.

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⁺ 2.x (Kir2.x) family members. The order of inhibition for all three drugs was Kir2.3 > Kir2.1 ∼ Kir2.2. The onset of inhibition of Kir2.x current by these compounds was slow (T_1/2 ∼ 6 min) and only partially recovered after washout (∼30%). 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 (PIP₂). Inhibition of Kir2.3 currents was significantly reduced by a single point mutation of I213L, which enhances Kir2.3 interaction with membrane PIP₂. Pretreatment with PIP₂ significantly decreased the inhibition induced by tamoxifen, 4-hydroxytamoxifen, and raloxifene on Kir2.3 channels. Pretreatment with spermine (100 μM) decreased the inhibitory effect of tamoxifen on Kir2.1, probably by strengthening the channel's interaction with PIP₂. In cat atrial and ventricular myocytes, 3 μM 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 PIP₂.

Hypercholesterolemia is a well-known risk factor for cardiovascular disease. In the heart, activation of K_ACh mediates the vagal (parasympathetic) negative chronotropic effect on heart rate. Yet, the effect of cholesterol on K_ACh is unknown. Here we show that cholesterol plays a critical role in modulating K_ACh currents (I_K,ACh) in atrial cardiomyocytes. Specifically, cholesterol enrichment of rabbit atrial cardiomyocytes led to enhanced channel activity while cholesterol depletion suppressed I_K,ACh. Moreover, a high-cholesterol diet resulted in up to 3-fold increase in I_K,ACh in rodents. In accordance, elevated currents were observed in Xenopus oocytes expressing the Kir3.1/Kir3.4 heteromer that underlies I_K,ACh. Furthermore, our data suggest that cholesterol affects I_K,ACh via a mechanism which is independent of both PI(4,5)P₂ and Gβγ. Interestingly, the effect of cholesterol on I_K,ACh is opposite to its effect on I_K1 in atrial myocytes. The latter are suppressed by cholesterol enrichment and by high-cholesterol diet, and facilitated following cholesterol depletion. These findings establish that cholesterol plays a critical role in modulating I_K,ACh in atrial cardiomyocytes via a mechanism independent of the channel's major modulators.