Two different mechanisms of disinhibition produced by GABAA receptor mutations linked to epilepsy in humans - PubMed (original) (raw)

Two different mechanisms of disinhibition produced by GABAA receptor mutations linked to epilepsy in humans

Matt T Bianchi et al. J Neurosci. 2002.

Abstract

The first mutations of the GABA(A) receptor channel linked to familial epilepsy in humans were reported recently (Baulac et al., 2001; Wallace et al., 2001). Preliminary functional analysis of alpha1beta2gamma2 GABA(A) receptors expressed in Xenopus oocytes suggested that the gamma2 subunit R43Q mutation abolished current enhancement by the benzodiazepine, diazepam, and that the gamma2 subunit K289M mutation decreased current amplitudes. We used single-channel recording and concentration jump techniques applied to outside out patches to evaluate the impact of these mutations on GABA(A) receptor channel function of the highly conserved rat ortholog subunits expressed in human embryonic kidney cells. When coexpressed with alpha1 and beta3 subunits, no differences were observed between wild-type and mutant GABA(A) receptor current activation rates or rates or extent of desensitization during prolonged (400 msec) GABA application (1 mm). Although deactivation after brief (5 msec) or prolonged (400 msec) GABA application was unaltered by the R43Q mutation, deactivation (a correlate of IPSC duration) was accelerated for the K289M mutation. Faster deactivation was likely a consequence of altered gating, because single-channel openings had shorter mean duration. Interestingly, the R43Q mutation did not alter diazepam potentiation. It did, however, substantially decrease current amplitude, which was not caused by decreased single-channel conductance or open time, suggesting reduced surface expression of functional receptors. The two gamma2 subunit mutations likely produce disinhibition and familial epilepsy by distinct mechanisms, suggesting that maintenance of neuronal inhibition depends not only on the peak amplitude of IPSCs, but also on their time course.

PubMed Disclaimer

Figures

Fig. 1.

Fig. 1.

Sensitivity to GABA and diazepam.A, The EC50 for GABA was determined by whole-cell responses to increasing GABA concentration, normalized to the peak current amplitude in each cell. Cells were voltage clamped at –10 to –50 mV, and data were obtained from three cells for each mutation. The dotted line is the concentration–response of wild-type α1β3γ2L GABAAreceptors (Bianchi et al., 2001). B, Diazepam sensitivity was determined by coapplication of 1 μ

m

diazepam with 2 μ

m

GABA (∼EC20) to whole cells expressing α1β3γ2L (top), α1β3γ2L(K289M), or α1β3γ2L(R43Q) GABAAreceptors. C, Diazepam enhancement is shown as the percentage of control responses (averagepeak current before and after diazepam coapplication). Neither mutation significantly affected modulation of GABA-evoked currents by diazepam. D, Representative currents are shown for two human α1β3γ2L(R43Q) GABAA receptor isoforms. The left pair of traces indicated the response of receptors containing a γ2L subunit with amino acid sequence corresponding to the original published γ2L sequence, whereas the_right pair_ of traces indicated the response of receptors containing a γ2L subunit with amino acid sequence corresponding to that of the rat γ2L subunit (see Materials and Methods). For both pairs of traces, the left current was evoked with 10 μ

m

GABA (solid bar), and the right current was evoked with coapplied 1 μ

m

diazepam (hatched bar) in the same cell. Similar results were obtained in five cells.

Fig. 2.

Fig. 2.

Macroscopic kinetic properties. A, Representative current traces obtained from wild-type or mutated receptors during 400 msec jumps into 1 m

m

GABA. Time scale of top trace applies to all three traces.B1_–_C2, Neither the fast (B1) nor the slow (C1) time constant of desensitization, nor their relative contributions (B2,C2) were significantly altered by the mutations.D, Current activation rate, as indicated by the 10–90% rise time of the current, was not significantly altered by the mutations. E, Peak current amplitudes were significantly smaller for α1β3γ2L(R43Q) GABAA receptors. *p < 0.01. F, Current deactivation after removal of GABA was significantly faster for α1β3γ2L(K289M) GABAA receptors. *p < 0.001. Data were obtained from 8–13 patches.

Fig. 3.

Fig. 3.

Deactivation after brief GABA pulses.A, Representative currents illustrate deactivation rates of α1β3γ2L (A1), α1β3γ2L(K289M) (A2), and α1β3γ2L(R43Q) (A3) GABAA receptors in response to brief (<5 msec) pulses of GABA (1 m

m

). Scale bars apply to all three_solid traces_. The solid trace in_A3_ is expanded 10-fold (gray trace) for comparison of deactivation current time course.B, Weighted time constants of deactivation (see Materials and Methods) are shown for wild-type and mutated channels. Deactivation was significantly faster for α1β3γ2L(K289M) GABAA receptors (hatched bar). *p < 0.05. α1β3γ2L (R43Q) GABAAreceptor deactivation (solid bar) was not different than that of wild-type receptors (gray bar). Data were obtained from 9–13 patches for each isoform.

Fig. 4.

Fig. 4.

Paired pulse inhibition. Pairs of 5 msec GABA (1 m

m

) pulses were delivered to outside-out patches at interpulse intervals of 30, 100, 300, and 1000 msec. A, Representative currents from wild-type and mutated GABAAreceptors. B, Summary plot showing the relative amplitude of the second pulse of each pair, for each four interpulse intervals. Less inhibition was observed for α1β3γ2L(K289M) GABAA receptors for each interpulse interval.

Fig. 5.

Fig. 5.

Single-channel analysis. Single-channel records obtained in patches held at −75 mV in the presence of 1 m

m

GABA from α1β3γ2L (A), α1β3γ2L(R43Q) (B), and α1β3γ2L(K289M) (C) GABAA receptors. A portion of the_top trace_ in each pair (indicated by the open bar) is expanded below that trace. Calibration bars apply to all three panels. Openings are downward. Similar results were observed from three α1β3γ2L, five α1β3γ2L(R43Q), and seven α1β3γ2L(K289M) patches. D, Open duration histogram for α1β3γ2L(K289M) single channels. The distribution was best described by the sum of three exponential functions, with each exponential fit shown as a smooth curve. Although three functions were required, the relative contribution of the shortest open state (leftmost curve) was highest, indicating that most openings were brief in duration. The time constants were 0.33, 1.17, and 4.54 msec with relative areas 0.81, 0.17, and 0.02, respectively. Data were pooled from seven α1β3γ2L(K289M) patches.

Similar articles

Cited by

References

    1. Angelotti TP, Uhler MD, Macdonald RL. Assembly of GABAA receptor subunits: analysis of transient single-cell expression utilizing a fluorescent substrate/marker gene technique. J Neurosci. 1993;13:1418–1428. - PMC - PubMed
    1. Bianchi MT, Macdonald RL. Agonist trapping by GABAA receptor channels. J Neurosci. 2001a;21:9083–9091. - PMC - PubMed
    1. Bianchi MT, Macdonald RL. Mutation of the 9′ leucine in the GABAA receptor γ2L subunit produces an apparent decrease in desensitization by stabilizing open states without altering desensitized states. Neuropharmacology. 2001b;41:737–744. - PubMed
    1. Bianchi MT, Haas KF, Macdonald RL. Structural determinants of fast desensitization and desensitization-deactivation coupling in GABAA receptors. J Neurosci. 2001;21:1127–1136. - PMC - PubMed
    1. Baulac S, Huberfeld G, Gourfinkel-An I, Mitropoulou G, Beranger A, Prud'homee J-F, Baulac M, Brice A, Bruzzone R, LeGuern E. First genetic evidence of GABAA receptor dysfunction in epilepsy: a mutation in the γ2-subunit gene. Nat Genet. 2001;28:46–48. - PubMed

Publication types

MeSH terms

Substances

Grants and funding

LinkOut - more resources