Inhibition of recombinant L-type voltage-gated calcium channels by positive allosteric modulators of GABAA receptors.
Benzodiazepines (BDZs) depress neuronal excitability via positive allosteric modulation of inhibitory GABA(A) receptors (GABA(A)R). BDZs and other positive GABA(A)R modulators, including barbiturates, ethanol, and neurosteroids, can also inhibit L-type voltage-gated calcium channels (L-VGCCs), which could contribute to reduced neuronal excitability. Because neuronal L-VGCC function is up-regulated after long-term GABA(A)R modulator exposure, an interaction with L-VGCCs may also play a role in physical dependence. The current studies assessed the effects of BDZs (diazepam, flurazepam, and desalkylflurazepam), allopregnanolone, pentobarbital, and ethanol on whole-cell Ba(2+) currents through recombinant neuronal Ca(v)1.2 and Ca(v)1.3 L-VGCCs expressed with beta(3) and alpha(2)delta-1 in HEK293T cells. Allopregnanolone was the most potent inhibitor (IC(50), approximately 10 muM), followed by BDZs (IC(50), approximately 50 muM), pentobarbital (IC(50), 0.3-1 mM), and ethanol (IC(50), approximately 300 mM). Ca(v)1.3 channels were less sensitive to pentobarbital inhibition than Ca(v)1.2 channels, similar to dihydropyridine (DHP) L-VGCC antagonists. All GABA(A)R modulators induced a negative shift in the steady-state inactivation curve of Ca(v)1.3 channels, but only BDZs and pentobarbital induced a negative shift in Ca(v)1.2 channel inactivation. Mutation of the high-affinity DHP binding site (T1039Y and Q1043M) in Ca(v)1.2 channels reduced pentobarbital potency. Despite the structural similarity between benzothiazepines and BDZs, mutation of an amino acid important for diltiazem potency (I1150A) did not affect diazepam potency. Although L-VGCC inhibition by BDZs occurred at concentrations that are possibly too high to be clinically relevant and is not likely to play a role in the up-regulation of L-VGCCs during long-term treatment, pentobarbital and ethanol inhibited L-VGCCs at clinically relevant concentrations.
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Benzodiazepines pentobarbital dihydropyridine DHP BDZs pentobarbital BDZs DHP pentobarbital benzothiazepines BDZs amino acid diltiazem diazepam BDZs BDZs pentobarbital barbiturates ethanol neurosteroids calcium BDZs diazepam flurazepam desalkylflurazepam allopregnanolone pentobarbital ethanol Ba(2+) Allopregnanolone BDZs pentobarbital ethanol calcium Ca(v)1.3 GABA(A) receptors Ca(v)1.2 L-VGCC GABA(A)R GABA(A)R Ca(v)1.3 Ca(v)1.2 high-affinity DHP binding site T1039Y Q1043M Ca(v)1.2 GABA(A)R I1150A L-VGCC L-VGCCs L-VGCCs L-type voltage-gated calcium channels L-VGCCs L-VGCC GABA(A)R L-VGCCs Ca(v)1.2 Ca(v)1.3 L-VGCCs GABAA receptors L-type voltage-gated calcium channels