Nifedipine Inhibition of High-Voltage Activated Calcium Channel Currents in Cerebral Artery Myocytes Is Influenced by Extracellular Divalent Cations.

Fei Wang,George C Wellman,Masayo Koide

doi:10.3389/fphys.2017.00210

Abstract

Voltage-dependent calcium channels (VDCCs) play an essential role in regulating cerebral artery diameter and it is widely appreciated that the L-type VDCC, CaV1.2, encoded by the CACNA1C gene, is a principal Ca2+ entry pathway in vascular myocytes. However, electrophysiological studies using 10 mM extracellular barium ([Ba2+]o) as a charge carrier have shown that ~20% of VDCC currents in cerebral artery myocytes are insensitive to 1,4-dihydropyridine (1,4-DHP) L-type VDDC inhibitors such as nifedipine. Here, we investigated the hypothesis that the concentration of extracellular divalent cations can influence nifedipine inhibition of VDCC currents. Whole-cell VDCC membrane currents were obtained from freshly isolated rat cerebral artery myocytes in extracellular solutions containing Ba2+ and/or Ca2+. In the absence of [Ca2+]o, both nifedipine-sensitive and -insensitive calcium currents were observed in 10 mM [Ba2+]o. However, VDCC currents were abolished by nifedipine when using a combination of 10 mM [Ba2+]o and 100 μM [Ca2+]o. VDCC currents were also completely inhibited by nifedipine in either 2 mM [Ba2+]o or 2 mM [Ca2+]o. The biophysical characteristics of all recorded VDCC currents were consistent with properties of a high-voltage activated VDCC, such as CaV1.2. Further, VDCC currents recorded in 10 mM [Ba2+]o ± 100 μM [Ca2+]o or 2 mM [Ba2+]o exhibited similar sensitivity to the benzothiazepine L-type VDCC blocker, diltiazem, with complete current inhibition at 100 μM. These data suggest that nifedipine inhibition is influenced by both Ca2+ binding to an external site(s) on these channels and surface charge effects related to extracellular divalent cations. In sum, this work demonstrates that the extracellular environment can profoundly impact VDCC current measurements.

Highlights

Members of the voltage-dependent calcium channel (VDCC) family are involved in a multitude of physiological processes throughout the body
Using 10 mM [Ba2+]o as a charge carrier, VDCC currents were elicited in rat cerebral artery myocytes by a series of
To examine the impact of [Ca2+]o on VDCC currents when 10 mM [Ba2+]o is used as the primary charge carrier, the above voltage-step protocol was repeated using myocytes bathed in a combination of 10 mM [Ba2+]o and 100 μM [Ca2+]o (Figures 1D–F)

Summary

Introduction

Members of the voltage-dependent calcium channel (VDCC) family are involved in a multitude of physiological processes throughout the body. It is widely appreciated that the L-type VDCC, CaV1.2, encoded by the gene CACNA1C, represents an important extracellular calcium ([Ca2+]o) entry pathway involved in the regulation of bulk cytosolic Ca2+ and contraction of vascular smooth muscle, including cerebral artery myocytes (Moosmang et al, 2003; Nystoriak et al, 2009). Yamada et al, using porcine coronary artery preparations, found that Ca2+ is required for high affinity binding of 1,4-DHPs to CaV1.2, whereas Ba2+ had the opposite effect, i.e., inhibition of ligand/channel binding. Because Ba2+ permeates several types of VDCCs more effectively than Ca2+, Ba2+ has commonly been used as a charge carrier during electrophysiological recordings to enhance the amplitude of VDCC membrane currents in cells having low channel number

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