TY - JOUR
T1 - Hydrophobic anions potently and uncompetitively antagonize GABA A receptor function in the absence of a conventional binding site
AU - Chisari, M.
AU - Wu, K.
AU - Zorumski, C. F.
AU - Mennerick, S.
PY - 2011/9
Y1 - 2011/9
N2 - BACKGROUND AND PURPOSE A 'lock-and-key' binding site typically accounts for the effect of receptor antagonists. However, sulphated neurosteroids are potent non-competitive antagonists of GABA A receptors without a clear structure-activity relationship. To gain new insights, we tested two structurally unrelated hydrophobic anions with superficially similar properties to sulphated neurosteroids. EXPERIMENTAL APPROACH We used voltage-clamp techniques in Xenopus oocytes and hippocampal neurons to characterize dipicrylamine (DPA) and tetraphenylborate (TPB), compounds previously used to probe membrane structure and voltage-gated ion channel function. KEY RESULTS Both DPA and TPB potently antagonized GABA A receptors. DPA exhibited an IC 50 near 60 nM at half-maximal GABA concentration and antagonism with features indistinguishable from pregnenolone sulphate antagonism, including sensitivity to a point mutation in transmembrane domain 2 of the α1 subunit. Bovine serum albumin, which scavenges free membrane-associated DPA, accelerated both capacitance offset and antagonism washout. Membrane interactions and antagonism were explored using the voltage-dependent movement of DPA between membrane leaflets. Washout of DPA antagonism was strongly voltage-dependent, paralleling DPA membrane loss, although steady-state antagonism lacked voltage dependence. At antagonist concentrations, DPA failed to affect inhibitory post-synaptic current (IPSC) amplitude or decay, but DPA accelerated pharmacologically prolonged IPSCs. CONCLUSIONS AND IMPLICATIONS Neurosteroid-like GABA A receptor antagonism appears to lacks a conventional binding site. These features highlight key roles of membrane interactions in antagonism. Because its membrane mobility can be controlled, DPA may be a useful probe of GABA A receptors, but its effects on excitability via GABA A receptors raise caveats for its use in monitoring neuronal activity.
AB - BACKGROUND AND PURPOSE A 'lock-and-key' binding site typically accounts for the effect of receptor antagonists. However, sulphated neurosteroids are potent non-competitive antagonists of GABA A receptors without a clear structure-activity relationship. To gain new insights, we tested two structurally unrelated hydrophobic anions with superficially similar properties to sulphated neurosteroids. EXPERIMENTAL APPROACH We used voltage-clamp techniques in Xenopus oocytes and hippocampal neurons to characterize dipicrylamine (DPA) and tetraphenylborate (TPB), compounds previously used to probe membrane structure and voltage-gated ion channel function. KEY RESULTS Both DPA and TPB potently antagonized GABA A receptors. DPA exhibited an IC 50 near 60 nM at half-maximal GABA concentration and antagonism with features indistinguishable from pregnenolone sulphate antagonism, including sensitivity to a point mutation in transmembrane domain 2 of the α1 subunit. Bovine serum albumin, which scavenges free membrane-associated DPA, accelerated both capacitance offset and antagonism washout. Membrane interactions and antagonism were explored using the voltage-dependent movement of DPA between membrane leaflets. Washout of DPA antagonism was strongly voltage-dependent, paralleling DPA membrane loss, although steady-state antagonism lacked voltage dependence. At antagonist concentrations, DPA failed to affect inhibitory post-synaptic current (IPSC) amplitude or decay, but DPA accelerated pharmacologically prolonged IPSCs. CONCLUSIONS AND IMPLICATIONS Neurosteroid-like GABA A receptor antagonism appears to lacks a conventional binding site. These features highlight key roles of membrane interactions in antagonism. Because its membrane mobility can be controlled, DPA may be a useful probe of GABA A receptors, but its effects on excitability via GABA A receptors raise caveats for its use in monitoring neuronal activity.
KW - GABA receptors
KW - antagonist
KW - hippocampal
KW - membrane capacitance
KW - voltage sensor
UR - http://www.scopus.com/inward/record.url?scp=80052213062&partnerID=8YFLogxK
U2 - 10.1111/j.1476-5381.2011.01396.x
DO - 10.1111/j.1476-5381.2011.01396.x
M3 - Article
C2 - 21457224
AN - SCOPUS:80052213062
SN - 0007-1188
VL - 164
SP - 667
EP - 680
JO - British Journal of Pharmacology
JF - British Journal of Pharmacology
IS - 2 B
ER -