Comparative Effects of Methylmercury and Hg on Human Neuronal N- and R-Type High-Voltage Activated Calcium Channels Transiently Expressed in Human Embryonic Kidney 293 Cells

Expression cDNA clones of 1B-1 or 1E-3 subunits coding for human neuronal N(Cav2.2) or R-subtype (Cav2.3) Ca 2 channels, respectively, was combined with 2-b and 3-a Ca 2 channel subunits, and transfected into human embryonic kidney cells for transient expression to determine whether specific types of neuronal voltage-sensitive Ca channels are affected differentially by methylmercury (MeHg) and Hg . For both Ca channel subtypes, MeHg (0.125–5.0 M) or Hg (0.1–5 M) caused a timeand concentration-dependent reduction of current. MeHg caused an initial, rapid component and a subsequent more gradual component of inhibition. The rapid component of block was completed between 100 and 150 s after beginning treatment. At 0.125 to 1.25 M, MeHg caused a more gradual decline in current. Apparent IC50 values were 1.3 and 1.1 M for MeHg, and 2.2 and 0.7 M for Hg on Nand R-types, respectively. For N-type current, effects of Hg were initially greater on the peak current than on the sustained current remaining at the end of a test pulse; subsequently, Hg blocked both components of current. For R-type current, Hg affected peak and sustained current approximately equally. Kinetics of inactivation also seemed to be affected by Hg in cells expressing N-type but not R-type current. Washing with MeHg-free solution could not reverse effects of MeHg on either type of current. The effect of Hg on Nbut not R-type current was partially reversed by Hg -free wash solution. Therefore, different types of Ca channels have differential susceptibility to neurotoxic mercurials even when expressed in the same cell type. Voltage-sensitive Ca channels play crucial roles in a number of cellular functions, including neurotransmitter release, gene expression, growth cone elongation, and dendritic action potential generation (Catterall, 1998, 2000). Various Ca channelopathies resulting in neuronal or neuromuscular disorders are caused by mutations in genes coding for Ca channel subunits (for review, see Meir and Dolphin, 2002). At least six distinct subtypes of Ca channels (L, N, T, P, Q, and R) have already been identified based on their differential biophysical, molecular biological, and pharmacological properties (Tsien et al., 1995). Neuronal Ca channels contain four subunits: 1, , 2, and . The 1 subunit is the pore-forming, voltage-sensing, and ligand-binding component. cDNAs for at least seven distinct subunits for high-voltage activated Ca channels: 1A-1F, 1S, have been cloned. Four different subunits and two different 2 subunits regulate assembly and modulate the kinetic parameters of the channel. The presence of several isoforms and splice variants further complicates the functional expression characteristics and classification of high-voltage activated Ca channels (Brust et al., 1993; DeWaard and Campbell, 1995; McEnery et al., 1998; Pan and Lipscombe, 2000). Cells typically coexpress several types of Ca channels, often with similar subcellular localization, providing a highly regulated degree of control over Ca -dependent cell functions, but confounding analyses of the properties of distinct Ca channel subtypes in isolation. Because of their portal location within the plasma membrane, Ca channels are potentially susceptible to the actions of a number of polyvalent heavy metal-type toxicants and serve as entry paths into the cell for heavy metals (Kiss and Osipenko, 1994; Atchison, 2003). This study was supported by National Institutes of Health Grants R01ES03299 and R01ES05822 (to W.D.A.). A preliminary report of these findings was presented at the 2001 Annual Meeting of the Society of Toxicology in San Francisco, CA and published in Toxicologist 60:185. 1 R.K.H. and S.-Q.P. contributed equally to this study. Article, publication date, and citation information can be found at http://jpet.aspetjournals.org. DOI: 10.1124/jpet.103.049429. ABBREVIATIONS: MeHg, methylmercury; IBa, Ba 2 current; HEK, human embryonic kidney; GFP, green fluorescent protein; V1/2, voltage for half-maximal activation. 0022-3565/03/3063-1129–1136$7.00 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 306, No. 3 Copyright © 2003 by The American Society for Pharmacology and Experimental Therapeutics 49429/1086503 JPET 306:1129–1136, 2003 Printed in U.S.A. 1129 at A PE T Jornals on Sptem er 6, 2017 jpet.asjournals.org D ow nladed from Because of the crucial roles that Ca channels play in key cellular functions, toxicant effects on Ca channels could have significant deleterious consequences for neuronal function. Methylmercury (MeHg) and inorganic mercury (Hg ) are environmental neurotoxicants that differ chemically in ionic charge, ionic radii, and lipophilicity. Together, these factors can impact the manner in which these mercurials affect a given cellular function. Neurotoxic mercurials act on a number of cellular targets. In several neuronal systems, cellular effects of MeHg and Hg are similar, yet distinct (Atchison et al., 1986; Hare and Atchison, 1992; Hewett and Atchison, 1992; Yuan and Atchison, 1994). The exact mechanisms by which these mercurials exert neurotoxicity are not known with certainty. Disruption of function of voltage-sensitive Ca channels is a prominent effect of acute exposure to low concentrations of both MeHg (Shafer and Atchison, 1991; Leonhardt et al., 1996; Sirois and Atchison, 1996, 2000; Shafer, 1998) and Hg (Büsselberg et al., 1991; Weisenberg et al., 1995). MeHg blocks Ba currents (IBa) carried through multiple subtypes of Ca channels in primary cultures of cerebellar granule cells and in rat pheochromocytoma (PC12) cells (Shafer and Atchison, 1991; Sirois and Atchison, 2000). Hg also alters function of several types of Ca channels at low micromolar concentrations (Büsselberg et al., 1994; Leonhardt et al., 1996; Szucs et al., 1997). However, the actions of mercurials on Ca channels may be more complex than mere block of function. In PC12 cells, very low concentrations of Hg increase amplitude of current carried through voltage-sensitive Ca channels (Rossi et al., 1993), whereas in cerebellar granule cells and NG108-15 cells, MeHg causes an increase in fura-2 fluorescence, which is dependent, at least in part, on extracellular Ca , and which is delayed by nifedipine, -conotoxin GVIA, and Ni (Hare and Atchison, 1995; Marty and Atchison, 1997). Moreover, treatment of rodents with Ca channel blockers prevents the toxic effects of MeHg (Sakamoto et al., 1996), and Ca channel blockers delay the onset of cerebellar granule cell death with MeHg (Marty and Atchison, 1997; Gasso et al., 2001). Finally, in cells lacking Ca channels, the onset of intracellular action of MeHg is delayed, suggesting that Ca channels provide a path of entry for MeHg into the cell (Edwards et al., 2002). Therefore, mercurials seem to interact with voltage-sensitive Ca channels in a complex manner. Because of the numerous and important roles that voltagesensitive Ca channels play in neuronal function, disruption of function of voltage-sensitive Ca channels may be a significant contributory factor in mercurial-induced neurotoxicity. There are few published reports comparing the effects of different mercurials on function of voltage-sensitive Ca channels (Hewett and Atchison, 1992; Szucs et al., 1997; Schirrmacher et al., 1998), and no comparative study on the effects of these two forms of mercury on defined types of voltage-sensitive Ca channels exists. The goal of the present study was to determine whether specific types of voltage-sensitive Ca channels were affected differentially by MeHg or Hg . We compared the effect of MeHg and Hg on N-(Cav2.2) and R-(Cav2.3) types of voltage-sensitive Ca channels expressed using cDNA copies of their genes transferred into human embryonic kidney cells (HEK293). These cells are nonexcitable and commonly used for heterologous expression of membrane proteins, including voltage-dependent Ca channels (Williams et al., 1994; Perez-Garcia et al., 1995; Quefurth et al., 1998). Expression cDNA clones of 1B-1 or 1E-3 subunit were combined with 2-b and 3-a Ca 2 channel subunits of human neuronal origin for transient expression of Nand R-subtypes, respectively, of high-voltage activated Ca channels. Jellyfish green fluorescent protein (GFP) was used as a cotrans-