Assessing use-dependent inhibition of the cardiac Na(+/-) current (I(Na)) in the PatchXpress automated patch clamp.
BACKGROUND
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.


CONCLUSIONS
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.
Penniman
Jacob R
JR
Safety and Exploratory Pharmacology, Safety Assessment, Merck Research Laboratories West Point, PA 19486, USA.
Kim
David C
DC
Salata
Joseph J
JJ
Imredy
John P
JP
D000200:Action Potentials / Q000187:drug effects; D000818:Animals; D004305:Dose-Response Relationship, Drug; D005424:Flecainide / Q000494:pharmacology; D006168:Guinea Pigs; D057809:HEK293 Cells; D006801:Humans; D008012:Lidocaine / Q000494:pharmacology; D008801:Mexiletine / Q000494:pharmacology; D009124:Muscle Proteins / Q000037:antagonists & inhibitors* / Q000378:metabolism; D009206:Myocardium / Q000378:metabolism*; D032383:Myocytes, Cardiac / Q000187:drug effects / Q000502:physiology*; D062554:NAV1.5 Voltage-Gated Sodium Channel; D018408:Patch-Clamp Techniques; D026941:Sodium Channel Blockers / Q000494:pharmacology*; D015222:Sodium Channels / Q000378:metabolism
D009124:Muscle Proteins; D062554:NAV1.5 Voltage-Gated Sodium Channel; C568320:SCN5A protein, human; D026941:Sodium Channel Blockers; D015222:Sodium Channels; D008801:Mexiletine; D008012:Lidocaine; D005424:Flecainide