John P. Imredy

Assessing use-dependent inhibition of the cardiac Na± current (INa) in the PatchXpress automated patch clamp

INTRODUCTION The cardiac Na+ current (I(Na)) underlies the rapid depolarization of the cardiac myocyte, and block of the current slows cardiac conduction and increases the risk of ventricular arrhythmia. A feature of Na+ channel block termed use-dependence is important to the assessment of blocking potency. We developed a robust automated patch clamp assay to rapidly and routinely assess the use-dependent block of I(Na) by drug candidates. The assay clarifies whether drug candidates block more potently at increased heart rates and provides a quantitative score of use-dependence. METHODS A use-dependent cardiac I(Na) assay was implemented on the PatchXpress 7000A, an automated whole-cell patch clamp device, using a HEK cell line stably expressing the human cardiac Na+ channel, Na(V)1.5. Stable recordings lasting up to 30 minutes were achieved by selection of holding potential (-100 mV) as well as an appropriate osmotic gradient to prevent time-dependent loss of cell capacitance and current. The final protocol allows evaluation of I(Na) inhibition at three pulsing rates at three test concentrations for each recorded cell. RESULTS IC(50) values obtained for three standard I(Na) blockers lidocaine, mexiletine, and flecainide, at pulsing frequencies of 0.2 Hz, 1 Hz, and 3 Hz, were compared to IC(50) values obtained with conventional pipette patch clamp of the Na(V)1.5 cell line and of guinea pig cardiac myocytes using matched voltage protocols and pulsing rates. Absolute potencies were well correlated only under conditions of matched holding potential and fell within an approximately three-fold window. While absolute potencies could vary widely with holding potential, the fold increases in potency with increases in pulsing rates were less prone to variation of the holding potential. DISCUSSION Use-dependence of cardiac Na+ channel block can be rapidly assessed in the PatchXpress platform and quantified at early stages of drug development to guide lead optimization.

Generation and Characterization of a Cell Line with Inducible Expression of Cav3.2 (T-Type) Channels

Establishment of stable cell lines that constitutively express Ca(2+) channels at high density and that are useful for in vitro studies may be complicated by problems with seal quality and duration during whole-cell patch-clamp electrophysiology. The current studies describe the generation and characterization of cells that express the human alpha1H T-type Ca(2+) channel under the control of a tetracycline-inducible expression system. Western blot and immunostaining studies revealed that expression of the alpha1H protein occurred only in the presence of tetracycline. Using the whole-cell patch-clamp method, the cells displayed peak inward currents of 1.15 +/- 0.14 nA in response to voltage-clamp steps. The T-type Ca(2+) current was inhibited by the T-type Ca(2+) channel antagonist, mibefradil, with an IC(50) of 160 nM. This cell line, with inducible channel expression, sealed with longer duration during whole-cell patch-clamp recording when compared with a cell line that constitutively expresses the alpha1H Ca(2+) channel. Ca(2+) influx through this channel could also be detected after the addition of extracellular Ca(2+). The amount of Ca(2+) influx was dependent on the [Ca](o) with an EC(50) of 4 mM. The Ca(2+) influx was also inhibited by mibefradil with a potency (IC(50) = 183 nM) similar to that observed in the voltage-clamp studies. Overall, this inducible alpha1H Ca(2+) channel-expressing cell line is useful for the study of human T-type Ca(2+) channel function, and offers advantages over a similar cell line that constitutively expresses the channel.

Interaction between amiodarone and hepatitis-C virus nucleotide inhibitors in human induced pluripotent stem cell-derived cardiomyocytes and HEK-293 Cav1.2 over-expressing cells

Several clinical cases of severe bradyarrhythmias have been reported upon co-administration of the Hepatitis-C NS5B Nucleotide Polymerase Inhibitor (HCV-NI) direct-acting antiviral agent, sofosbuvir (SOF), and the Class-III anti-arrhythmic amiodarone (AMIO). We model the cardiac drug-drug interaction (DDI) between AMIO and SOF, and between AMIO and a closely-related SOF analog, MNI-1 (Merck Nucleotide Inhibitor #1), in functional assays of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs), to provide mechanistic insights into recently reported clinical cases. AMIO co-applied with SOF or MNI-1 increased beating rate or field potential (FP) rate and decreased impedance (IMP) and Ca(2+) transient amplitudes in hiPSC-CM syncytia. This action resembled that of Ca(2+) channel blockers (CCBs) in the model, but CCBs did not substitute for AMIO in the DDI. AMIO analog dronedarone (DRON) did not substitute for, but competed with AMIO in the DDI. Ryanodine and thapsigargin, decreasing intracellular Ca(2+) stores, and SEA-0400, a Na(+)/Ca(2+) exchanger-1 (NCX1) inhibitor, partially antagonized or suppressed DDI effects. Other agents affecting FP rate only exerted additive or subtractive effects, commensurate with their individual effects. We also describe an interaction between AMIO and MNI-1 on Cav1.2 ion channels in an over-expressing HEK-293 cell line. MNI-1 enhanced Cav1.2 channel inhibition by AMIO, but did not affect inhibition of Cav1.2 by DRON, verapamil, nifedipine, or diltiazem. Our data in hiPSC-CMs indicate that HCV-NI agents such as SOF and MNI-1 interact with key intracellular Ca(2+)-handling mechanisms. Additional study in a Cav1.2 HEK-293 cell-line suggests that HCV-NIs potentiate the inhibitory action of AMIO on L-type Ca(2+) channels.

Cardiac drug-drug interaction between HCV-NS5B pronucleotide inhibitors and amiodarone is determined by their specific diastereochemistry

Severe bradycardia/bradyarrhythmia following coadministration of the HCV-NS5B prodrug sofosbuvir with amiodarone was recently reported. Our previous preclinical in vivo experiments demonstrated that only certain HCV-NS5B prodrugs elicit bradycardia when combined with amiodarone. In this study, we evaluate the impact of HCV-NS5B prodrug phosphoramidate diastereochemistry (D-/L-alanine, R-/S-phosphoryl) in vitro and in vivo. Co-applied with amiodarone, L-ala,SP prodrugs increased beating rate and decreased beat amplitude in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs), but D-ala,RP produgs, including MK-3682, did not. Stereochemical selectivity on emerging bradycardia was confirmed in vivo. Diastereomer pairs entered cells equally well, and there was no difference in intracellular accumulation of L-ala,SP metabolites ± amiodarone, but no D-ala,RP metabolites were detected. Cathepsin A (CatA) inhibitors attenuated L-ala,SP prodrug metabolite formation, yet exacerbated L-ala,SP + amiodarone effects, implicating the prodrugs in these effects. Experiments indicate that pharmacological effects and metabolic conversion to UTP analog are L-ala,SP prodrug-dependent in cardiomyocytes.

Interaction of the BKCa channel gating ring with dendrotoxins

Two classes of small homologous basic proteins, mamba snake dendrotoxins (DTX) and bovine pancreatic trypsin inhibitor (BPTI), block the large conductance Ca2+-activated K+ channel (BKCa, KCa1.1) by production of discrete subconductance events when added to the intracellular side of the membrane. This toxin-channel interaction is unlikely to be pharmacologically relevant to the action of mamba venom, but as a fortuitous ligand-protein interaction, it has certain biophysical implications for the mechanism of BKCa channel gating. In this work we examined the subconductance behavior of 9 natural dendrotoxin homologs and 6 charge neutralization mutants of δ-dendrotoxin in the context of current structural information on the intracellular gating ring domain of the BKCa channel. Calculation of an electrostatic surface map of the BKCa gating ring based on the Poisson-Boltzmann equation reveals a predominantly electronegative surface due to an abundance of solvent-accessible side chains of negatively charged amino acids. Available structure-activity information suggests that cationic DTX/BPTI molecules bind by electrostatic attraction to site(s) on the gating ring located in or near the cytoplasmic side portals where the inactivation ball peptide of the β2 subunit enters to block the channel. Such an interaction may decrease the apparent unitary conductance by altering the dynamic balance of open versus closed states of BKCa channel activation gating.