Neuroactive steroids have multiple actions to potentiate GABAA receptors

Abstract

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 alpha1 beta2 gamma2L subunits. Activity was elicited with a half-maximal (50 microM) concentration of GABA. Two steroids were studied in detail: ACN ((3alpha,5alpha,17beta)-3-hydroxyandrostane-17-carbonitrile) and B285 ((3alpha,5beta,17beta)-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 three 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 microM. The differences in the actions of these two 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 two functional actions. The closed time distributions also showed three components in the absence of steroid. The rate of occurrence of the two 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 two independent mechanisms, perhaps the sites and mechanisms associated with actions on open times. The presence of 1 microM 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 gamma2 subunit, but not the alpha1 or beta2 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 is more than one distinguishable steroid recognition site on the GABAA receptor.

Figure 1. Single channel currents elicited by 50 μm GABA in the absence or presence of steroids

Sample clusters are shown in the left column, while the distributions of open times in clusters are shown on the right. Superimposed on the histograms are the fits for three exponential components (thin lines) and the sum of all three (heavy lines). ACN at 10 nm has little effect, while 1 μm ACN increases both the fraction and the mean duration of the long duration component (note the shift in the right-most component fitted to the data). In contrast, 1 μm B285 increases the proportion of long duration openings (note increased area under the right-most component) but not the mean duration. Finally, 30 μm B285 increases both the fraction and the mean duration of the long duration open time component. The structures of ACN and B285 are shown at the bottom of the figure. Note that ACN has the same structure as allopregnanolone, except for the presence of a carbonitrile at the 17-position instead of a methylketone. B285 differs in two places: the absence of a methyl group at the 18 position, and altered ring fusion at the 5 position.

Figure 2. Effects of steroids on mean durations

The arithmetic mean open times (A), closed times (B) and calculated P_open (C) in clusters elicited by 50 μm GABA in the presence of various concentrations of ACN (•) or B285 (▵) are shown. The curves show the fit of the equation Y([steroid]) =Y₀+ (Y_max–Y₀)[steroid]/([steroid]+ EC₅₀) for data obtained in the presence of ACN (continuous lines) and B285 (dashed lines). (The continuous line in B shows the mean closed time in the presence of ACN; no fit was performed to these data.) The dotted lines show the mean values for data obtained using 50 μm GABA in the absence of steroid. Values for the fit parameters are given in Table 1.

Figure 3. Effects of steroids on prevalence and duration of open times

The fitted mean durations (top row) and fraction of total openings (bottom row) of the open time components are shown (ACN: • B285: ▵). Data are shown for the brief duration component (OT₁, panels A and B), the intermediate duration component (OT₂, panels C and D) and the long duration component (OT₃, panels E and F). The lines are as described in the legend to Fig. 2; if no fitted curve is shown then no fit was attempted. Values for fit parameters are given in Table 1.

Figure 4. Effects of steroids on the rates of entry into closed channel states

The rates of occurrence for various closed time components (per second of open time) are shown. The left column shows data obtained in the presence of ACN, and the right column shows data in the presence of B285. In each panel, the point plotted at the left shows data obtained using 50 μm GABA in the absence of steroid, while the dotted lines show the mean values obtained over a range of GABA concentrations in the absence of steroid. The fitted curves were obtained as described in the legend to Table 1. The top panels (A and D) show the sum of all closing rates (▾) and the apparent short duration desensitized state (□). (Note that the short duration desensitized state was not resolved in the presence of B285, nor in the presence of 50 μm GABA alone.) The middle panels (B and E) show the rate of occurrence of the CT₃ component. The bottom panels (C and F) show the two brief duration components, CT₂ (○) and CT₁ (♦). Note that ACN has more profound effects to decrease the rates of occurrence of CT₁, CT₂ and CT₃, and, accordingly, a more profound effect on the overall closing rate. However, B285 does decrease the rate of occurrence of CT₃ with a similar EC₅₀, while only the highest concentrations of B285 affect the rates of occurrence of CT₁ and CT₂. Parameters for the fitted curves are given in Table 1.

Figure 5. Lack of effect of 1 μm ACN on the channel effective opening rate

The channel effective opening rate is plotted against the concentration of GABA, in the absence (⋄) and presence (•) of 1 μm ACN. The data were fitted with the equation β′([GABA]) =β([GABA]ⁿ)/{[GABA]ⁿ+ EC₅₀ⁿ}. The values fitted were: GABA alone, β= 1883 ± 686 s⁻¹, EC₅₀= 359 ± 162 μm, n = 1.7 ± 0.2 and GABA + 1 μm ACN, β= 1475 ± 947 s⁻¹, EC₅₀= 374 ± 365 μm, n = 1.4 ± 0.3.

Figure 6. The presence of 1 μm B285 reduces effects of ACN on the long duration open time component

The mean duration of the long duration open time component (OT₃) is shown in A, while the fraction of the total openings which belonged to that component is shown in B, for data obtained in the presence of 1 μm B285 plus the indicated concentrations of ACN (▾). The continuous lines are the fits to data obtained with ACN alone (see Fig. 3), the dashed lines show mean values obtained in the presence of 1 μm B285 alone and the dotted lines show the mean values for data obtained using 50 μm GABA in the absence of steroid. No attempt was made to fit the data.

Figure 7. The presence of 1 μm B285 reduces effects of ACN on the rates of entering closed channel states

The rates of occurrence of the various closed time components in the presence of 1 μm B285 plus the indicated concentration of ACN are shown. The curves show the fits to data obtained in the presence of ACN alone (see Fig. 4), the dashed lines show values in the presence of 1 μm B285 alone and the dotted lines show mean values obtained in the presence of GABA alone. No attempt was made to fit the data. Note that the presence of 1 μm B285 reduces the actions of ACN.

Figure 8. Single channel currents elicited by 50 μm GABA from GABA_A receptors containing mutated subunits

Clusters of activity are shown, from receptors containing (α1–ρ)β2γ2 subunits elicited by 50 μm GABA (top row) or GABA plus 1 μm ACN (2nd row). Note that the sojourns in open states are prolonged in the presence of ACN. Clusters from receptors containing α1β2(γ2–ρ) subunits elicited by 50 μm GABA (3rd row) or GABA plus 1 μm ACN (bottom row). Note that open sojourns are not obviously prolonged, although closed state sojourns appear to be reduced in duration.

Figure 9. The relative changes produced by ACN or pentobarbital on receptors containing wild-type or mutated subunits

A, mean duration of the OT₃ component in the presence of 1 μm ACN, normalized to the value in the presence of GABA alone, and similar data in the presence of 40 μm pentobarbital (PB). Data were obtained from receptors containing α1β2γ2 (•), (α1–ρ)β2γ2 (▴), α1(β2–ρ)γ2 (▀), α1β2(γ2–ρ) (□) and (α1–ρ)(β2–ρ)(γ2–ρ) (▵) subunits. B, data for the rates of entry to the CT₁, CT₂, CT₃ states and the sums of those rates. Data are shown for receptors in the presence of 1 μm ACN (•, wild-type; ○, α1β2(γ2–ρ)) or 40 μm PB (▴, wild-type; ▵, α1β2(γ2–ρ)); normalized to the value in the presence of GABA alone. Note that the ordinate in panel A is linear, while that in panel B is logarithmic. Data are means ±s.d. Non-normalized data and results of statistical tests are shown in Tables 2 and 3.

Figure 10. A hypothetical kinetic scheme which accounts for many of the actions of neuroactive steroids on GABA_A receptor function

A receptor with a closed channel is designated by C, an agonist molecule by A, a receptor with an open channel as O, and the brief duration closed states which underlie the CT₁ and CT₂ components as G₁ and G₁′. The actions of steroids are hypothesized to occur by actions to increase the rate constant for going from A₂O₂ to A₂O₃ (site A effect), to decrease the rate for going from A₂O₁ to A₂C (site A effect), and to decrease the rate for going from A₂O₃ to A₂O₂ (site B effect).