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Controlling ion-channel desensitization
CELLECTRICON
created. They patch-clamped whole cells also an important factor. As the expoIon channels control the transport of that were engineered to express a type sure time increased, the population of ions into and out of cells, so underof rat GABAA receptor. Via a cyclic, receptors in a cell gradually separated standing how these channels behave is into one of two EC50 values. The EC50 critical for the development of pharmascanning patch-clamp protocol, the inceutical therapies. In this issue of Anavestigators recorded changes in the values differed by as much as a factor of lytical Chemistry (pp 7947–7953), Owe whole-cell current as they moved the 17 for GABA and by nearly a factor of Orwar, Daniel Granfeldt, and colleagues cells across an increasing or decreasing 40 for -alanine. at Chalmers University of Technology concentration gradient of either GABA Granfeldt says the data suggest that and Cellectricon (both in Sweden) disor -alanine, both of which are activaprior “experiences” of the receptors cuss how they could control the desentors of the GABAA receptor. must be taken into account when the sitization of ligand-gated ion chaneffects of activators or inhibitors are nels and change their responses to studied. “You need to know what activators or inhibitors. The findyour receptor population has [preings demonstrate how experimental viously] gone through, because you designs could potentially skew data can get different effects from the and distort the determination of drug same substance at the same concenefficacy. tration,” he says. But he does point Ligand-gated ion channels, such out, “The effect we describe is deas the GABAA (-aminobutyric pendent on the specific GABA receptor subunits/subtypes present in acid A) receptor, are activated the cell. Different subunits can give when an agonist binds to them and quite different desensitization betriggers a response. The channels havior and may therefore produce open, but they soon become dedifferent results.” sensitized—they just sit in a ligandVariations in responses weren’t bound state and don’t conduct any just limited to agonists—antagocurrent. Researchers have recently nists were also found to trigger difbeen paying more attention to the A patch-clamped cell expressing ligand-gated ion chanfering responses in receptors dedesensitized states, because these nels moves across a concentration gradient of agonist pending on prior exposure. Orwar states change the conducting prop- or antagonist generated by a microfluidic device. and colleagues tested a competitive erties of the channels. However, GABAA receptor antagonist called bicucomplex kinetics hamper the analysis of To get a handle on the extent of dedesensitization; for instance, convenculline. They filled the 16 microchansensitization, the investigators looked at tional patch-clamp measurements can nels, alternating between buffer and inthe EC50 value in the dose–response only detect the open, conducting states creasing amounts of bicuculline against curves. (The EC50 value represents the of the channels. a constant concentration of GABA. concentration of agonist needed to proBut Orwar and colleagues had a miBecause they were studying an antagvoke a response halfway between the crofluidic device at their disposal. The onist, the investigators looked at the baseline and maximum responses.) device creates patterned laminar flows in Orwar and colleagues discovered that IC50 value, the concentration required an open chamber by flowing solutions the GABAA receptors displayed high or for 50% inhibition of the receptors. When out of 16 closely spaced microchannels they looked at the blocking efficacy of low EC50 values depending on the ex(Anal. Chem. 2004, 76, 4968– 4976). bicuculline on high- and low-EC50 popposure time and whether the agonist “We knew that desensitization was really concentration gradient increased or deulations of the GABAA receptor, Orwar important for shaping the communicacreased. The differences in EC50 values and colleagues found a difference of a tion between neuronal cells,” says Gran- were due to receptors assembling in factor of ~5 in blocking efficacy. This feldt. “We thought we could use the finding suggested that the potency of an varying desensitized states. The effect chip we had in a novel way and get to inhibitor is influenced by the activation had very little to do with the affinities the desensitization problem that way.” history of the receptors. Computer modof GABA and -alanine for the recepOrwar and colleagues filled the 16 eling of the ligand-gated ion channel detors and appeared to be an intrinsic microchannels, alternating between a sensitization states produced predictions property of the GABAA receptors. buffer and an agonist solution, so that a similar to the experimental results. a The length of time a receptor popuconcentration gradient of agonist was —Rajendrani Mukhopadhyay lation was exposed to the agonist was D E C E M B E R 1 , 2 0 0 6 / A N A LY T I C A L C H E M I S T R Y
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