José A. Sánchez-Chapula

Molecular determinants of voltage-dependent human ether-a-go-go related gene (HERG) K+ channel block.

The structural determinants for the voltage-dependent block of ion channels are poorly understood. Here we investigate the voltage-dependent block of wild-type and mutant human ether-a-go-go related gene (HERG) K+ channels by the antimalarial compound chloroquine. The block of wild-type HERG channels expressed in Xenopusoocytes was enhanced as the membrane potential was progressively depolarized. The IC50 was 8.4 ± 0.9 μmwhen assessed during 4-s voltage clamp pulses to 0 mV. Chloroquine also slowed the apparent rate of HERG deactivation, reflecting the inability of drug-bound channels to close. Mutation to alanine of aromatic residues (Tyr-652 or Phe-656) located in the S6 domain of HERG greatly reduced the potency of channel block by chloroquine (IC50 > 1 mm at 0 mV). However, mutation of Tyr-652 also altered the voltage dependence of the block. In contrast to wild-type HERG, block of Y652A HERG channels was diminished by progressive membrane depolarization, and complete relief from block was observed at +40 mV. HERG channel block was voltage-independent when the hydroxyl group of Tyr-652 was removed by mutating the residue to Phe. Together these findings indicate a critical role for Tyr-652 in voltage-dependent block of HERG channels. Molecular modeling was used to define energy-minimized dockings of chloroquine to the central cavity of HERG. Our experimental findings and modeling suggest that chloroquine preferentially blocks open HERG channels by cation-π and π-stacking interactions with Tyr-652 and Phe-656 of multiple subunits.

Molecular genetic and functional association of Brugada and early repolarization syndromes with S422L missense mutation in KCNJ8.

BACKGROUND Adenosine triphosphate (ATP)-sensitive potassium cardiac channels consist of inward-rectifying channel subunits Kir6.1 or Kir6.2 (encoded by KCNJ8 or KCNJ11) and the sulfonylurea receptor subunits SUR2A (encoded by ABCC9). OBJECTIVE To examine the association of mutations in KCNJ8 with Brugada syndrome (BrS) and early repolarization syndrome (ERS) and to elucidate the mechanism underlying the gain of function of ATP-sensitive potassium channel current. METHODS Direct sequencing of KCNJ8 and other candidate genes was performed on 204 BrS and ERS probands and family members. Whole-cell and inside-out patch-clamp methods were used to study mutated channels expressed in TSA201 cells. RESULTS The same missense mutation, p.Ser422Leu (c.1265C>T) in KCNJ8, was identified in 3 BrS and 1 ERS probands but was absent in 430 alleles from ethnically matched healthy controls. Additional genetic variants included CACNB2b-D601E. Whole-cell patch-clamp studies showed a 2-fold gain of function of glibenclamide-sensitive ATP-sensitive potassium channel current when KCNJ8-S422L was coexpressed with SUR2A-wild type. Inside-out patch-clamp evaluation yielded a significantly greater half maximal inhibitory concentration for ATP in the mutant channels (785.5 ± 2 vs 38.4 ± 3 μM; n = 5; P <.01), pointing to incomplete closing of the ATP-sensitive potassium channels under normoxic conditions. Patients with a CACNB2b-D601E polymorphism displayed longer QT/corrected QT intervals, likely owing to their effect to induce an increase in L-type calcium channel current (I(Ca-L)). CONCLUSIONS Our results support the hypothesis that KCNJ8 is a susceptibility gene for BrS and ERS and point to S422L as a possible hotspot mutation. Our findings suggest that the S422L-induced gain of function in ATP-sensitive potassium channel current is due to reduced sensitivity to intracellular ATP.

The molecular basis of chloroquine block of the inward rectifier Kir2.1 channel

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 IK1. 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 block IK1 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.

Effects of diabetic cardiomyopathy on regional electrophysiologic characteristics of rat ventricle

Aims/hypothesis. To identify the possible causes of the lengthening of the action potential duration described in patients affected by diabetes mellitus.¶Methods. We studied the effects of streptozotocin-induced diabetes on the current density of the repolarising potassium currents Ito, IK, Iss and IK1 in enzymatically isolated myocytes from three different regions of rat heart: total right ventricle, subepicardium at the apex of the left ventricle and subendocardium at the base of the left ventricle.¶Results. No changes in IK1 were found due to diabetes, but there was a uniform decrease in Ito (50 %) and Iss (40 %) current densities in the three regions. In contrast, IK diminished unevenly, with the greatest decrease in the subendocardium at the base of the left ventricle (48 %), followed by the subepicardium at the apex of the left ventricle (32 %) and right ventricle (10 %).¶Conclusion/interpretation. These findings suggest the existence of regional differences in ion channel expression associated with diabetes. The decrease of these repolarising currents could account for the lengthening of action potential and the consequent change in the Q-T interval of the ECG observed in diabetic rats. [Diabetologia (2000) 43: 101–109]

DIFFERENCES IN REGIONAL DISTRIBUTION OF K+ CURRENT DENSITIES IN RAT VENTRICLE

The objective of the present work is to study the ionic mechanisms for the regional differences in action potential duration in rat ventricle. This regional diversity has been related to differences in the regional distribution of some potassium currents in several species. Single cells were obtained by enzymatic dispersion of tissue segments from rat ventricular muscle. Whole cell voltage-clamp methods were used to identify the K+ currents involved in action potential repolarisation in the different regions. 4-Aminopiridine, TEA and voltage protocols were used to isolate the following potassium currents: transient outward, Ito, delayed rectifier, Ik, and sustained current, Iss. In the present work, we have studied the distribution of these three repolarising currents, and that of the inward rectifier, Ikl, in the free wall of the right ventricle, the subepicardium of the apex of the left ventricle and in the subendocardium of the base of the left ventricle. Action potential duration was longer in the left than in the right ventricle, and in the former it was longer in the subendocardium of the base than in the subepicardium of the apex. The main difference was in the phase 1, suggesting the implication of Ito. This was confirmed with voltage-clamp experiments. In conclusion, this work shows that Ito current density is higher in the regions with the shorter action potential, whereas there are no differences in the regional distribution of Ik, Iss or Ikl.

ABCC9 is a novel Brugada and early repolarization syndrome susceptibility gene.

BACKGROUND Genetic defects in KCNJ8, encoding the Kir6.1 subunit of the ATP-sensitive K(+) channel (I(K-ATP)), have previously been associated with early repolarization (ERS) and Brugada (BrS) syndromes. Here we test the hypothesis that genetic variants in ABCC9, encoding the ATP-binding cassette transporter of IK-ATP (SUR2A), are also associated with both BrS and ERS. METHODS AND RESULTS Direct sequencing of all ERS/BrS susceptibility genes was performed on 150 probands and family members. Whole-cell and inside-out patch-clamp methods were used to characterize mutant channels expressed in TSA201-cells. Eight ABCC9 mutations were uncovered in 11 male BrS probands. Four probands, diagnosed with ERS, carried a highly-conserved mutation, V734I-ABCC9. Functional expression of the V734I variant yielded a Mg-ATP IC₅₀ that was 5-fold that of wild-type (WT). An 18-y/o male with global ERS inherited an SCN5A-E1784K mutation from his mother, who displayed long QT intervals, and S1402C-ABCC9 mutation from his father, who displayed an ER pattern. ABCC9-S1402C likewise caused a gain of function of IK-ATP with a shift of ATP IC₅₀ from 8.5 ± 2 mM to 13.4 ± 5 μM (p<0.05). The SCN5A mutation reduced peak INa to 39% of WT (p<0.01), shifted steady-state inactivation by -18.0 mV (p<0.01) and increased late I(Na) from 0.14% to 2.01% of peak I(Na) (p<0.01). CONCLUSION Our study is the first to identify ABCC9 as a susceptibility gene for ERS and BrS. Our findings also suggest that a gain-of-function in I(K-ATP) when coupled with a loss-of-function in SCN5A may underlie type 3 ERS, which is associated with a severe arrhythmic phenotype.

Effects of bupivacaine on membrane currents of guinea-pig ventricular myocytes

The effects of bupivacaine on the membrane currents of single guinea-pig ventricular myocytes were investigated using the whole-cell patch-clamp technique. Bupivacaine decreased the inward calcium current in a concentration-dependent way at concentrations of 10 microM and higher. Bupivacaine also decreased the delayed outward current without modifying the inward-rectifying potassium current. From these results it can be concluded that bupivacaine, at concentrations lower than 10 microM, does not exert its negative inotropic effect by decreasing the calcium current. This mechanism may play a role at higher concentrations of the drug.

Tamoxifen inhibits inward rectifier K+ 2.x family of inward rectifier channels by interfering with phosphatidylinositol 4,5-bisphosphate-channel interactions.

Tamoxifen, an estrogen receptor antagonist used in the treatment of breast cancer, inhibits the inward rectifier potassium current (IK1) 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 IK1. 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 (T1/2 ∼ 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 channels interaction with PIP2. In cat atrial and ventricular myocytes, 3 μM tamoxifen inhibited IK1, 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.

Mechanism of Block of Cardiac Transient Outward K+ Current (Ito) by Antidepressant Drugs

Imipramine, amitriptyline, mianserine, maprotiline, and trazodone are five widely used antidepressant drugs with different chemical structures. Imipramine and amitriptyline are tricyclics, mianserine and maprotiline are tetracyclics, and trazodone is a triazolopyridine derivative. We studied the effects of these drugs on the transient outward K+ current (I(to)) and the interaction mechanisms within the drug molecules and the channel-binding site. The transient outward K+ current is mainly responsible for action-potential repolarization in the rat ventricle, and all of the five drugs studied block I(to), but in different manners. Cyclic drugs block I(to) in the open state of the channel with very little block in the rested or inactivated states or both. Trazodone blocks the channel in a state-independent manner. From these results, we suggest that a relation exists between drug structure and preference for the different channel conformations.

Conformational changes in the M2 muscarinic receptor induced by membrane voltage and agonist binding

Non‐technical summary  Muscarinic receptors were recently shown to be modulated by membrane potential. Here, we show that membrane potential alters the binding of agonists in an agonist‐specific manner. Moreover, agonist binding results in agonist‐specific conformational changes in the muscarinic receptor, as measured by changes in the receptors response to voltage. Voltage‐dependent modulation of muscarinic receptors has important consequences for cellular signalling in excitable tissues and implications for cardiovascular drug development.