GABA_A receptors (GABA_ARs) are the primary inhibitory neurotransmitter receptors throughout the central nervous system. Genetic mutations causing their dysfunction are related to a broad spectrum of human disorders such as epilepsy, neurodevelopment and intellectual disability, autism spectrum disorder, schizophrenia, and depression. GABA_ARs are also important drug targets for anxiolytics, anticonvulsants, antidepressants, and anesthetics. Despite significant progress in understanding their three-dimensional structure, a critical gap remains in determining the molecular basis for channel gating. We recently identified mutations in the M2-M3 linkers that suggest linker flexibility has asymmetric subunit-specific correlations with channel opening. Here we use non-canonical amino acids (ncAAs) to investigate the role of main-chain H-hydrogen bonds (H-bonds) that may stabilize the M2-M3 linkers. We show that a single main-chain H-bond within the β2 subunit M2-M3 linker inhibits pore opening and is required to keep the unliganded channel closed. Furthermore, breaking this H-bond accounts for approximately one third of the energy used to open the channel during activation by GABA. In contrast, the analogous H-bond in the α1 subunit has no effect on gating. Our molecular simulations support the idea that channel opening involves the state-dependent breakage/disruption of a specific main-chain H-bond within the β2 subunit M2-M3 linker.