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.

Keywords: CaV3.2 T-Channel; Dorsal Root Ganglion; Ethanol; Phorbol 12-Myristate 13-Acetate; Protein Kinase C.

Fig. 1

Ethanol selectively inhibits Ca_V3.2 channels recorded from human embryonic kidney (HEK) 293 cell lines. (A) Representative current inactivation traces of Ca_V3.2 channels recorded before and at ten minutes after the bath application of 100 mM ethanol. Current inactivation was elicited at −50 mV from holding potentials starting from −135 to −60 mV for one second. (B) 100 mM ethanol induced a strong hyperpolarizing shift in the steady-state inactivation curve in the whole cell current (fitted with Boltzmann equation). (C, D) 100 mM ethanol had no significant effect on steady-state inactivation curves in currents recorded from HEK cells expressing Ca_V3.1 and Ca_V3.3 channels. (E) V₅₀ derived from the inactivation curve was significantly shifted to a more hyperpolarized membrane potential after application of 100 mM ethanol in the cells expressing Ca_V3.2 channels. (n =11, p<0.01, **, paired t-test) but it did not change in Ca_V3.1 or Ca_V3.3 currents. (F) 100 mM ethanol decreased current density in Ca_V3.2 channels but had no effect in Ca_V3.1 and Ca_V3.3 channels (n=10, p<0.05, *, paired t-test). (G) Representative current activation traces were elicited from −95 mV to 0 mV in 6 mV increments from a holding potential of −135 mV for one second. (H) Average I-V relationship before and during the application of 100 mM ethanol. The I-V curve was plotted with normalized peak current against the command potentials. After 10 minutes application of ethanol in the bath, peak current reduced about 32% (n=7, p<0.001, paired t-test). (I) Steady-state activation was deduced from I-V curves presented in (H) and fitted with Boltzmann equation. V₅₀ Derived from activation curve showed no significant changes after application of 100 mM Ethanol. *p<0.05; **p<0.01.

Fig. 2

Biphasic, dose-dependent effect of ethanol on current density in Ca_V3.2 channels from HEK cell lines. (A) Representative traces and time course showing the effects of 100 mM ethanol on Ca_V3.2 channels (scale bar: 50 msec, 1 nA). Peak reduction was reached 10 minutes after applying ethanol. (Bi) 50, 100 and 200 mM ethanol significantly reduced current density (2-way ANOVA followed by Bonferroni post hoc test). (Bii) The percentage changes of normalized current density obtained between the ethanol and control current measured in the same cell showed only 100 and 200 mM ethanol were significantly different from control (1-way ANOVA followed by Dunnett's post hoc test). *p<0.05; **p<0.01;**** p<0.0001.

Fig. 3

PKC mediated ethanol effects on kinetics and current density of Ca_V3.2 currents recorded from HEK cell lines. Myristoylated Protein Kinase C (PKC) peptide inhibitor (MPI: 50 μM) was delivered through the pipette internal solution. (A) Representative inactivation Ca_V3.2 current traces recorded in the absence, presence of 100 mM ethanol in the recording chamber with the PKC inhibitor in the internal solution (the same protocol as was used in Fig.1). (B) 100 mM ethanol showed no hyperpolarizing shift in the steady-state inactivation curve in Ca_V3.2 channels with PKC inhibitor in the internal solution. (C) Ethanol significantly shifted V₅₀ to a more hyperpolarized membrane potential. MPI blocked ethanol effects. ((D,E,F) Ethanol produced significantly faster rise time, decay constant and reduction in current density, MPI blocked ethanol effects on decay constant. ((Gi) 50 mM ethanol produced a hyperpolarizing shift in the steady-state inactivation curve in Ca_V3.2 channels (fitted with Boltzmann equation). V₅₀ derived from the inactivation curve was significantly shifted to a more hyperpolarized membrane potential. (Gii) 50 mM ethanol showed no hyperpolarizing shift in the steady-state inactivation curve in Ca_V3.2 channels when 50 μM MPI was added in the internal solution. *p<0.05; **p<0.01;*** p<0.001.

Fig. 4

Lower (100 nM) and higher (1 μM) concentrations of phorbol 12-myristate 13-acetate (PMA) produce different effects on steady-state inactivation and current density of Ca_V3.2 channels from HEK cell lines. (A, C) Representative inactivation Ca_V3.2 current traces recorded in the absence, and ten minutes after application in the recording chamber of 100 nM, 1 μM PMA and wash out with ACSF (the same inactivation protocol as was used in Fig. 1). (B) 100 nM PMA induced a strong hyperpolarizing shift in steady-state inactivation curve in Ca_V3.2 channels. (D) 1 μM PMA had no effect on the inactivation curve. (E) 100 nM PMA significantly shifted V₅₀ to a more hyperpolarized membrane potential, but 1 μM PMA had no significant effect. (F) 100 nM PMA significantly reduced current density but 1 μM PMA increased current density. **p<0.01;**** p<0.0001.

Fig. 5

The effects of lower (100 nM) and higher (1 μM) concentrations of PMA on kinetic properties in Ca_V3.2 currents recorded from HEK cell lines. (A) 1 μM PMA significantly increased rise slope. (B) Both 100 nM PMA 1 μM PMA significantly reduced rise time. (C) 1 μM PMA produced a significantly faster decay constant that recovered upon washout. (D) 100 nM PMA significantly reduced charge. ((E) 50 μM MPI blocked hyperpolarizing shift in steady-state inactivation curve induced by 100 nM PMA. (F). 100 nM PMA induced a hyperpolarizing shift in steady-state inactivation curve, but no further shift was observed after adding 100 mM ethanol, demonstrating occlusion of 100 nM PMA on ethanol effects. *p<0.05;**p<0.01;**** p<0.0001.

Fig. 6

PKC mediated inhibition of ethanol on T current in dorsal root ganglion (DRG) neurons. (A) 100 mM ethanol produced a hyperpolarizing shift in the inactivation curve (the same protocol as was used in Fig 1). (B) There was no hyperpolarizing shift after application of 100 mM ethanol while the PKC peptide inhibitor (MPI: 50 μM) was in the internal solution. (C) Ethanol significantly shifted V₅₀ to a more hyperpolarized membrane potential, and it was blocked by MPI. (D) Ethanol significantly reduced current density. *p<0.05;***p<0.001.

Fig. 7

The effects of lower (100 nM) and higher (1μM) concentrations of PMA on T-type currentsin DRG neurons. (A) Representative T current traces recorded in the absence and ten minutes after application of 100 nM PMA in the recording chamber, and after washout with ACSF. 100 nM PMA reduced T current, which partially recovered upon washout with ACSF. (B) 100 nM PMA shifted the steady-state inactivation curve to a more hyperpolarized membrane potential. (C) 1 μM PMA increased T current amplitude, which recovered upon washout with ACSF. (D) 1 μM PMA had no effect on the steady-state inactivation curve. (E) 100 nM PMA significantly shifted V₅₀ to a more hyperpolarized membrane potential, but 1 μM PMA had no effect. (F) 100 nM PMA significantly reduced but 1 μM PMA significantly enhanced current density. (G) 1μM PMA significantly produced a faster decay constant. (H) 100 nM PMA significantly reduced charge.