Changes in Memory, Sedation, and Receptor Kinetics Imparted by the ß2-N265M and ß3-N265M GABAA Receptor Point Mutations.

Point mutations in the ß2 (N265S) and ß3 (N265M) subunits of γ-amino butyric acid type A receptors (GABAARs) that render them insensitive to the general anesthetics etomidate and propofol have been used to link modulation of ß2-GABAARs to sedation and ß3-GABAARs to surgical immobility. These mutations also alter GABA sensitivity, and mice carrying the ß3-N265M mutation have been reported to have impaired baseline memory. Here, we tested the effects of the ß2-N265M and ß3-N265M mutations on memory, movement, hotplate sensitivity, anxiety, etomidate-induced sedation, and intrinsic kinetics. We found that both ß2-N265M and ß3-N265M mice exhibited baseline deficits in the Context Preexposure Facilitation Effect learning paradigm. Exploratory activity was slightly greater in ß2-N265M mice, but there were no changes in either genotype in anxiety or hotplate sensitivity. ß2-N265M mice were highly resistant to etomidate-induced sedation, and heterozygous mice were partially resistant. In rapid solution exchange experiments, both mutations accelerated deactivation two- to three-fold compared to wild type receptors and prevented modulation by etomidate. This degree of change in the receptor deactivation rate is comparable to that produced by an amnestic dose of etomidate but in the opposite direction, indicating that intrinsic characteristics of GABAARs are optimally tuned under baseline conditions to support mnemonic function.

Isoflurane Potentiation of GABAA Receptors Is Reduced but Not Eliminated by the ß3(N265M) Mutation.

Background: Mice carrying the GABAA receptor ß3(N265M) point mutation, which renders receptors incorporating ß3-subunits insensitive to many general anesthetics, have been used experimentally to link modulation of different receptor subtypes to distinct behavioral endpoints. Remarkably, however, the effect of the mutation on the susceptibility to modulation by isoflurane (a standard reference agent for inhalational vapors) has never been tested directly. Therefore, we compared the modulation by isoflurane of expressed α5ß3(N265M)γ2L receptors with their wild type counterparts. Methods: Using whole-cell electrophysiological recording and rapid solution exchange techniques, we tested the effects of isoflurane at concentrations ranging from 80 µM to 320 µM on currents activated by 1 µM GABA. We measured drug modulation of wild-type α5ß3γ2L GABAA receptors and their counterparts harboring the ß3(N265M) mutation. Results: Currents elicited by GABA were enhanced two- to four-fold by isoflurane, in a concentration-dependent manner. Under the same conditions, receptors incorporating the ß3(N265M) mutation were enhanced by approximately 1.5- to two-fold; i.e., modulation by isoflurane was attenuated by approximately one-half. Direct activation by isoflurane was also present in mutant receptors but also attenuated. Conclusions: In contrast to the complete insensitivity of ß3(N265M) mutant receptors to etomidate and propofol, the mutation has only a partial effect on receptor modulation by isoflurane. Therefore, the persistence of isoflurane effects in mutant mice does not exclude a possible contribution of ß3-GABAA receptors.

Flow characterization and patch clamp dose responses using jet microfluidics in a tubeless microfluidic device.

BACKGROUND: Surface tension passive pumping is a way to actuate flow without the need for pumps, tubing or valves by using the pressure inside small drop to move liquid via a microfluidic channel. These types of tubeless devices have typically been used in cell biology. Herein we present the use of tubeless devices as a fluid exchange platform for patch clamp electrophysiology. NEW METHOD: Inertia from high-speed droplets and jets is used to create flow and perform on-the-fly mixing of solutions. These are then flowed over GABA transfected HEK cells under patch in order to perform a dose response analysis. RESULTS: TIRF imaging and electrical recordings are used to study the fluid exchange properties of the microfluidic device, resulting in 0-90% fluid exchange times of hundreds of milliseconds. COMSOL is used to model flow and fluid exchange within the device. Patch-clamping experiments show the ability to use high-speed passive pumping and its derivatives for studying peak dose responses, but not for studying ion channel kinetics. COMPARISON WITH EXISTING METHOD(S): Our system results in fluid exchange times slower than when using a standard 12-barrel application system and is not as stable as traditional methods, but it offers a new platform with added functionality. CONCLUSIONS: Surface tension passive pumping and tubeless devices can be used in a limited fashion for electrophysiology. Users may obtain peak dose responses but the system, in its current form, is not capable of fluid exchange fast enough to study the kinetics of most ion channels.

Enhancement of α5-containing γ-aminobutyric acid type A receptors by the nonimmobilizer 1,2-dichlorohexafluorocyclobutane (F6) is abolished by the ß3(N265M) mutation.

BACKGROUND: Modulation of γ-aminobutyric acid type A receptors (GABAARs) by general anesthetics may contribute to their ability to produce amnesia. Receptors containing α5 subunits, which mediate tonic and slow synaptic inhibition, are co-localized with ß3 and γ2 subunits in dendritic layers of the hippocampus and are sensitive to low (amnestic) concentrations of anesthetics. Because α5 and ß3 subunits influence performance in hippocampus-dependent learning tasks in the presence and absence of general anesthetics, and the experimental inhaled drug 1,2-dichlorohexafluorocyclobutane (F6) impairs hippocampus-dependent learning, we hypothesized that F6 would modulate receptors that incorporate α5 and ß3 subunits. We hypothesized further that the ß3(N265M) mutation, which controls receptor modulation by general anesthetics, would similarly influence modulation by F6. METHODS: Using whole-cell electrophysiologic recording techniques, we tested the effects of F6 at concentrations ranging from 4 to 16 µM on receptors expressed in human embryonic kidney 293 cells. We measured drug modulation of wild-type α5ß3 and α5ß3γ2L GABAARs and receptors harboring the ß3(N265M) mutation. We also tested the effects of F6 on α1ß2γ2L receptors, which were reported previously to be insensitive to this drug when expressed in Xenopus oocytes. RESULTS: F6 enhanced the responses of wild-type α5ß3γ2L but not α1ß2γ2L receptors to low concentrations of GABA in a concentration-dependent manner. Receptors that incorporated the mutant ß3(N265M) subunit were insensitive to F6. When applied together with a high concentration of GABA, F6 blocked currents through α5ß3 but not α5ß3γ2L receptors. F6 did not alter deactivation of α5ß3γ2L receptors after brief high- concentration pulses of GABA. CONCLUSIONS: The nonimmobilizer F6 modulates GABAARs in a manner that depends on subunit composition and mode of receptor activation by GABA, supporting a possible role for α5-containing receptors in suppression of learning and memory by F6. Furthermore, common structural requirements indicate that similar molecular mechanisms may be responsible for the enhancing effects of F6 and conventional general anesthetics.

m-Azipropofol (AziPm) a photoactive analogue of the intravenous general anesthetic propofol.

Propofol is the most commonly used sedative-hypnotic drug for noxious procedures, yet the molecular targets underlying either its beneficial or toxic effects remain uncertain. In order to determine targets and thereby mechanisms of propofol, we have synthesized a photoactivateable analogue by substituting an alkyldiazirinyl moiety for one of the isopropyl arms but in the meta position. m-Azipropofol retains the physical, biochemical, GABA(A) receptor modulatory, and in vivo activity of propofol and photoadducts to amino acid residues in known propofol binding sites in natural proteins. Using either mass spectrometry or radiolabeling, this reagent may be used to reveal sites and targets that underlie the mechanism of both the desirable and undesirable actions of this important clinical compound.