title: Ethanol inhibition of a T-type Ca²+ channel through activity of protein kinase C.
abstract: BACKGROUND
  T-type calcium channels (T-channels) are widely distributed in the central and peripheral nervous system, where they mediate calcium entry and regulate the intrinsic excitability of neurons. T-channels are dysregulated in response to alcohol administration and withdrawal. We therefore investigated acute ethanol (EtOH) effects and the underlying mechanism of action in human embryonic kidney (HEK) 293 cell lines, as well as effects on native currents recorded from dorsal root ganglion (DRG) neurons cultured from Long-Evans rats.
  
  
  METHODS
  Whole-cell voltage-clamp recordings were performed at 32 to 34°C in both HEK cell lines and DRG neurons. The recordings were taken after a 10-minute application of EtOH or protein kinase C (PKC) activator (phorbol 12-myristate 13-acetate [PMA]).
  
  
  RESULTS
  We recorded T-type Ca²⁺ currents (T-currents) from 3 channel isoforms (CaV3.1, CaV3.2, and CaV3.3) before and during administration of EtOH. We found that only 1 isoform, CaV3.2, was significantly affected by EtOH. EtOH reduced current density as well as producing a hyperpolarizing shift in steady-state inactivation of both CaV3.2 currents from HEK 293 cell lines and in native T-currents from DRG neurons that are known to be enriched in CaV3.2. A myristoylated PKC peptide inhibitor (MPI) blocked the major EtOH effects, in both the cell lines and the DRG neurons. However, PMA effects were more complex. Lower concentration PMA (100 nM) replicated the major effects of EtOH, while higher concentration PMA (1 μM) did not, suggesting that the EtOH effects operate through activation of PKC and were mimicked by lower concentration of PMA.
  
  
  CONCLUSIONS
  EtOH primarily affects the CaV3.2 isoform of T-type Ca²⁺ channels acting through PKC, highlighting a novel target and mechanism for EtOH effects on excitable membranes.
authors.affiliation: Department of Neurobiology and Anatomy, Wake Forest University School of Medicine, Winston-Salem, NC 27157, USA.
authors.forename: Hong Qu
authors.initials: HQ
authors.lastname: Shan
authors.forename: James A
authors.initials: JA
authors.lastname: Hammarback
authors.forename: Dwayne W
authors.initials: DW
authors.lastname: Godwin
mesh_terms: D020747:Calcium Channels, T-Type / Q000378:metabolism*; D002492:Central Nervous System Depressants / Q000494:pharmacology*; D000431:Ethanol / Q000494:pharmacology*; D057809:HEK293 Cells; D006801:Humans; D009474:Neurons / Q000378:metabolism; D020033:Protein Isoforms / Q000037:antagonists & inhibitors / Q000378:metabolism; D011493:Protein Kinase C / Q000378:metabolism*
chemical_list: D020747:Calcium Channels, T-Type; D002492:Central Nervous System Depressants; D020033:Protein Isoforms; D000431:Ethanol; D011493:Protein Kinase C
keywords: CaV3.2 T-Channel; Dorsal Root Ganglion; Ethanol; Phorbol 12-Myristate 13-Acetate; Protein Kinase C