Ranolazine Blocks NaV1.5 Peak Current and Shear Sensitivity in Human Colonic Circular Smooth Muscle

Abstract

Introduction:SCN5A-encoded NaV1.5 is a voltage-gated mechanosensitive sodium channel expressed in circular layer smooth muscle cells (SMCs) and interstitial cells of Cajal of human small intestine. In human small intestinal smooth muscle, NaV1.5 contributes to resting membrane potential and slow waves. SCN5A mutations are found in 2-3% of patients with irritable bowel syndrome (IBS). The majority of these SCN5A mutations are associated with constipation predominant IBS. A Na+ current was previously described in colonic circular SMCs, and we recently identified SCN5A mRNA in human colonic circular muscle suggesting NaV1.5 is also expressed in the colon. Ranolazine is a clinically available NaV1.5 antagonist used in cardiac disease that also blocks NaV1.5 mechanosensitivity. Constipation is a common adverse effect of ranolazine. The aim of this study was to examine effect of ranolazine on Na+ current and shear sensitivity in human colonic circular SMCs. Methods: Whole cell inward currents were recorded from freshly dissociated human colonic circular SMCs with and without 50 μmol/L ranolazine in bath. Shear stress was induced by 10 mL/min flow of bath solution containing 0 or 50 μmol/L ranolazine. The distinct time and voltage dependent characteristics of fast activating Na+ current and slower activating Ca2+ current were used to separate the components of inward current. The voltage dependence of activation was fitted by a Boltzmann equation. Comparisons were made by paired t-test of means ±SEM. Results: In colonic circular SMCs, ranolazine (50 μmol/L) inhibited NaV1.5 peak currents by 43±9% (-104±33 to -58±20 pA/pF; n=6; P<0.05). Ranolazine shifted the half-point of the voltage dependence of activation (V1/2) from -37±1.7 to -43±1.4 mV (n=6; P<0.05). Mechanical stimulation by flow (shear stress) increased colonic SMC NaV1.5 peak current by 18±2% (−104±33 to −122±39 pA/pF; n=6; P<0.05). V1/2 was hyperpolarized with shear stress from -37±1.7 to -40±2.2 mV (n=6; P<0.05). Shear stress accelerated time to peak activation at -30 mV from 5.4±0.6 to 2.9±0.4 ms (n=5; P<0.05). Sensitivity to shear was blocked by ranolazine (-58±20 to -60±28 pA/pF; n=6; P=0.2). Ranolazine also blocked change in V1/2 and activation caused by shear (-43±1.4 mv to -43±1.1; n=6; P=0.7 for V1/2 and 2.9±0.4 to 4.3±1.4 ms, n=3; P<0.05 for activation time with ranolozine). Conclusion: Ranolazine blocked human colonic circular SMC Na+ currents and also blocked Na+ current shear sensitivity. These effects of ranolazine may help explain the gastrointestinal side effects of the drug and may be of clinical interest for the treatment of disorders such as diarrhea-predominant IBS. Supported by NIH DK 57266 and DK 57061.