Rapid kinetic properties of GABA(A) receptor channel currents.

Since the cloning of GABA$\sb{\rm A}$ receptor (GABAR) subunit families, whole-cell patch-clamp studies have provided extensive information about the biophysical and pharmacological properties of recombinant GABARs over a time scale of hundreds of milliseconds to several seconds. Yet, at GABAergic synapses, events that shape GABAR currents take place on a scale of $\mu$secs to tens of milliseconds. To understand the factors that shape synaptic GABAR currents, the biophysical and pharmacological properties of recombinant GABARs need to be examined on the time scale of synaptic events. To examine the time course of recombinant GABAR currents on such a rapid time scale, we have utilized a technique for rapidly applying GABA to outside-out membrane patches. First, we examined the contributions of the $\delta$ and $\gamma$ GABAR subunit families in shaping the rapid kinetics of recombinant GABAR currents, finding that $\delta$-containing GABARs activate more slowly, desensitize more slowly and less completely, and deactivate more rapidly than $\gamma$2L-containing GABARs. These macroscopic currents are shaped by the underlying pattern of activity of many single channels. Therefore, we examined GABA steady-state single-channel currents at a predicted synaptic GABA concentration. We utilized these rapid kinetic and single-channel data to develop more comprehensive kinetic models that predict the behavior of recombinant GABARs containing $\delta$ or $\gamma$2L GABAR subtypes. These models reconcile microscopic and macroscopic current kinetics and predict that differences in against unbinding rates, channel opening rates, and desensitization are responsible for the differences in $\delta$-containing and $\gamma$2L-containing GABAR kinetics. We used $\delta$-$\gamma$2L subunit subtype chimeras and site-specific mutagenesis to explore the structural bases behind these kinetic differences, localizing the determinants of gating and desensitization to a region that included the M1, the M1-M2 linker, and M2 domains. We examined the roles of different GABAR agonists and of the anticonvulsant agent, pentobarbital, in shaping the rapid kinetics of GABAR currents. These experiments, in conjunction with the insights gained from developing the kinetic models, enhance our understanding of the factors that are likely to be important in shaping the time course of GABAergic IPSCs.PhDAnimal PhysiologyBiological SciencesCellular biologyHealth and Environmental SciencesNeurosciencesPharmacologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/131432/2/9909896.pd