| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH |
|
|
|
|||||||
|
|||||||||
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Received April 14, 2004
Revised May 11, 2004
Accepted after revision May 14, 2004
1 Washington University in St Louis
* To whom correspondence should be addressed. E-mail: jhs{at}morpheus.wustl.edu.
The effects of neuroactive steroids on the function of GABAA receptors were studied using cell-attached records of single channel activity recorded from HEK293 cells transfected with 
1 
2 
2L subunits. Activity was elicited with a half-maximal (50 µM) concentration of GABA. Two steroids were studied in detail: ACN ((3,5,17)-3-hydroxyandrostane-17-carbonitrile) and B285 ((3,5,17)-3-hydroxy-18-norandrostane-17-carbonitrile). Four effects on channel activity were seen, two on open time distributions and two on closed times.
When clusters of openings were elicited in the absence of steroid, the open time distribution contained 3 components. ACN produced concentration dependent alterations in the open time distribution. The prevalence of the longest duration class of open times was increased from about 15% to about 40% (EC50 about 180 nM ACN), while the duration of the longest class increased from 7.4 ms to 27 ms (EC50 about 35 nM ACN). B285 also increased the prevalence of the longest duration open times (EC50 about 18 nM B285) but increased the duration only at concentrations close to 10 µM. The differences in the actions of these 2 steroids suggest that the effects on proportion and duration of the long duration open time component are produced by independent mechanisms and that there are separate recognition sites for the steroids which are associated with the 2 functional actions.
The closed time distributions also showed 3 components in the absence of steroid. The rate of occurrence of the 2 brief duration closed time components decreased with increasing ACN, with an EC50 of about 50 nM ACN. In contrast, B285 did not reduce the rate of occurrence of the brief closings until high concentrations were applied. However, both B285 and ACN reduced the rate of occurrence of the activation-related closed state selectively, with comparable IC50 concentrations (about 40 nM ACN, 20 nM B285). As in the case for action on open times these data suggest that there are two recognition sites and 2 independent mechanisms, perhaps the sites and mechanisms associated with actions on open times. The presence of 1 ?M ACN had no effect on the estimated channel opening rate or on the apparent affinity of the receptor for GABA.
Mutation of the carboxy terminus of the 2 subunit, but not the 1 or 2 subunits, abolished the ability of ACN to increase the duration of OT3 but had no effect on the reduction of the rate of occurrence of the activation-related closed state. These observations are also consistent with the idea that there are more than one distinguishable steroid recognition site on the GABAA receptor.
This article has been cited by other articles:
|
|
 |
|
  G. Akk, P. Li, J. Bracamontes, D. E. Reichert, D. F. Covey, and J. H. Steinbach Mutations of the GABA-A Receptor {alpha}1 Subunit M1 Domain Reveal Unexpected Complexity for Modulation by Neuroactive Steroids Mol. Pharmacol., September 1, 2008; 74(3): 614 - 627. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P. Li, H.-J. Shu, C. Wang, S. Mennerick, C. F. Zorumski, D. F. Covey, J. H. Steinbach, and G. Akk Neurosteroid migration to intracellular compartments reduces steroid concentration in the membrane and diminishes GABA-A receptor potentiation J. Physiol., November 1, 2007; 584(3): 789 - 800. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 E. W. Kelly, K. Solt, and D. E. Raines Volatile Aromatic Anesthetics Variably Impact Human {gamma}-Aminobutyric Acid Type A Receptor Function Anesth. Analg., November 1, 2007; 105(5): 1287 - 1292. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 W. Li, X. Jin, D. F. Covey, and J. H. Steinbach Neuroactive Steroids and Human Recombinant {rho}1 GABA Receptors J. Pharmacol. Exp. Ther., October 1, 2007; 323(1): 236 - 247. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. Li, J. Bracamontes, B. W. Katona, D. F. Covey, J. H. Steinbach, and G. Akk Natural and Enantiomeric Etiocholanolone Interact with Distinct Sites on the Rat {alpha}1beta2{gamma}2L GABAA Receptor Mol. Pharmacol., June 1, 2007; 71(6): 1582 - 1590. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
G. Akk, P. Li, B. D. Manion, A. S. Evers, and J. H. Steinbach Ethanol Modulates the Interaction of the Endogenous Neurosteroid Allopregnanolone with the {alpha}1beta2{gamma}2L GABAA Receptor Mol. Pharmacol., February 1, 2007; 71(2): 461 - 472. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. G. Hales, T. Z. Deeb, H. Tang, K. A. Bollan, D. P. King, S. J. Johnson, and C. N. Connolly An Asymmetric Contribution to {gamma}-Aminobutyric Type A Receptor Function of a Conserved Lysine within TM2-3 of {alpha}1, beta2, and {gamma}2 Subunits J. Biol. Chem., June 23, 2006; 281(25): 17034 - 17043. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. Li, D. F. Covey, J. H. Steinbach, and G. Akk Dual Potentiating and Inhibitory Actions of a Benz[e]indene Neurosteroid Analog on Recombinant {alpha}1beta2{gamma}2 GABAA Receptors Mol. Pharmacol., June 1, 2006; 69(6): 2015 - 2026. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
G. M. C. Lema and A. Auerbach Modes and models of GABAA receptor gating J. Physiol., April 1, 2006; 572(1): 183 - 200. [Abstract] [Full Text] [PDF]
|
|
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH |
