Roy E. Twyman

Neurosteroid regulation of GABAA receptor single-channel kinetic properties of mouse spinal cord neurons in culture.

1. Single‐channel kinetics of steroid enhancement of single gamma‐aminobutyric acidA (GABA) receptor currents obtained from somata of mouse spinal cord neurones in culture were investigated using the excised outside‐out patch‐clamp recording technique. GABA (2 microM) and GABA (2 microM) plus androsterone (5 alpha‐androstan‐3 alpha‐ol‐17‐one, AND, 10 nM‐10 microM) or pregnanolone (5 beta‐pregnan‐3 alpha‐ol‐20‐one, PRE, 100 nM‐10 microM) applied by pressure ejection from micropipettes evoked inward currents when patches were voltage clamped at ‐75 mV in symmetrical chloride solutions. Averaged GABA receptor currents were increased in the presence of the steroids. 2. GABA receptor currents were recorded with at least two conductance levels, a predominant or main‐conductance level of about 28 pS (which contributed 96% of the current evoked) and a minor or sub‐conductance level of about 20 pS. The current amplitudes of the two conductance levels were unchanged by the steroids. The gating (opening and closing) kinetics of both of the conductance levels were analysed. Findings for the main‐conductance level are summarized below. 3. Both steroids increased the average GABA receptor channel open duration. Consistent with the increased GABA receptor channel average open duration, the steroids shifted frequency histograms of GABA receptor channel open durations to longer durations. Three exponential functions were required to fit best the frequency histograms of GABA open durations, consistent with at least three kinetic open states of the main‐conductance level. Time constants obtained from the GABA receptor channel open‐duration frequency histograms were unchanged in the presence of the steroids. The basis for the increased average GABA receptor channel open durations by the steroids was due to an increased relative proportion of the two longer open‐duration time constants. The GABA receptor channel average open durations were increased by AND and PRE in a concentration‐dependent manner by shifting the proportion of openings to the longer open time constants. At a concentration of 10 microM, the prolongation of the average open duration was decreased, suggesting that the GABA receptor channel was blocked by these steroids. 4. GABA receptor channel opening frequency was increased and average channel‐closed duration was decreased by AND or PRE. Consistent with this, areas of the frequency histograms of channel closed durations were shifted to shorter durations. Closed frequency distributions were fitted best with five to six exponential functions, suggesting that the channel had multiple kinetic closed states. The three briefest time constants were not greatly altered by the steroids.(ABSTRACT TRUNCATED AT 400 WORDS)

Cyclic AMP-dependent protein kinase decreases GABAA receptor current in mouse spinal neurons

GABA, the major inhibitory neurotransmitter in the mammalian brain, binds to GABAA receptors, which form chloride ion channels. The predicted structure of the GABAA receptor places a consensus phosphorylation site for cAMP-dependent protein kinase (PKA) on an intracellular domain of the channel. Phosphorylation by various protein kinases has been shown to alter the activity of certain ligand- and voltage-gated ion channels. We have examined the role of phosphorylation by the catalytic subunit of PKA in the regulation of GABAA receptor channel function using whole-cell and excised outside-out patch-clamp techniques. Inclusion of the catalytic subunit of PKA in the recording pipettes significantly reduced GABA-evoked whole-cell and single-channel chloride currents. Both heat inactivation of PKA and addition of the specific protein kinase inhibitor peptide prevented the reduction of GABA-evoked currents by PKA. Neither mean channel open time nor channel conductance was affected by PKA. The reduction in GABA receptor current by PKA was primarily due to a reduction in channel opening frequency.

Kinetic properties of the GABAA receptor main conductance state of mouse spinal cord neurones in culture.

1. The kinetic properties of the main conductance state of gamma‐aminobutyric acidA (GABA) receptor channels from somata of mouse spinal cord neurones in cell culture were investigated using patch clamp techniques. 2. Whole‐cell GABA receptor currents increased in a concentration‐dependent manner from 0.5 to 5 microM. 3. Single‐channel currents were recorded with a main conductance state of 27.2 pS and a less frequent conductance state of 15.9 pS. The main conductance state opened singly and in bursts of several openings. 4. Mean open times of GABA receptor main conductance currents were increased and open‐time frequency histograms were shifted to longer times as GABA concentration was increased from 0.5 to 5 microM. Three exponential functions were required to fit the histograms at all GABA concentrations, suggesting that the channel opened into at least three open states (O1, O2 and O3). The three functions had the same time constants (1.0 +/‐ 0.2, 3.7 +/‐ 0.4 and 11.3 +/‐ 0.5 ms; mean +/‐ S.D.) at each concentration. The increase in long open times with concentration was due to a shift in relative frequency of occurrence of openings from the shortest (O1) to the two longest (O2 and O3) open states. 5. Closed‐time distributions of closures between main conductance state openings were fitted with multiple exponential functions, suggesting that the channel had several closed states. The two shortest time constants (0.24 +/‐ 0.03 and 2.0 +/‐ 0.3 ms) were concentration independent (0.5 to 5 microM). Three longer time constants decreased as concentration increased. 6. Bursts were defined as groups of openings surrounded by closures greater than a critical closed time (tc = 5 ms). Mean burst durations were increased and burst duration frequency histograms were shifted to longer times as GABA concentration was increased from 0.5 to 5 microM. Burst‐duration frequency histograms were best fitted with three exponential functions. The time constants were concentration independent and were 1.0 +/‐ 0.2, 5.5 +/‐ 0.2 and 29.8 +/‐ 1.6 ms. The increase in burst duration with concentration was due to a relative shift from short duration bursts to longer duration bursts. 7. The shortest burst time constant was similar to the shortest open time constant suggesting that there was a population of single openings of short duration. The two longest burst time constants were longer than the two longest open time constants, suggesting that the bursts from the two longest burst components were composed of two or more openings.(ABSTRACT TRUNCATED AT 400 WORDS)

Benzodiazepine and beta‐carboline regulation of single GABAA receptor channels of mouse spinal neurones in culture.

1. The effects of the benzodiazepine receptor agonist, diazepam (DZ), and the inverse agonist, methyl‐6,7‐dimethoxy‐4‐ethyl‐beta‐carboline‐3‐carboxylate (DMCM), on gamma‐aminobutyric acid (GABAA) receptor single channel currents were characterized. Outside‐out patches were obtained from somata of cultured mouse spinal cord neurones and voltage clamped at ‐75 mV (ECl = 0 mV). 2. GABA (2 microM) alone or with DZ (20‐1000 nM) or DMCM (20‐100 nM) was applied to patches by pressure ejection from blunt micropipettes. DZ enhanced GABAA receptor currents with an inverted U‐shaped concentration‐response curve. Mean steady‐state currents were increased by low concentrations of DZ (20‐50 nM). At higher concentrations of DZ, the enhancement was diminished. Mean steady‐state currents were decreased by DMCM at all concentrations. 3. GABAA receptor channels opened most frequently to a 27 pS main conductance level and less frequently to a 19 pS subconductance level. Neither DZ nor DMCM altered the proportion of time spent at either of the conductance levels. The kinetic properties of the main conductance level were studied. 4. Neither DZ nor DMCM altered the mean GABAA receptor channel open or burst durations. Sums of three exponential functions were required to fit best open and burst duration‐frequency histograms for GABA alone or with DZ or DMCM. No significant changes in the three time constants or areas of the three exponential functions for open or burst duration histograms were produced by DZ or DMCM. 5. With increasing concentrations of DZ up to 50 nM, GABA evoked an increased frequency of channel openings and bursts. With higher DZ concentrations, the magnitudes of the increase in channel opening and burst frequencies were reduced. At all concentrations of DMCM, GABA evoked a decreased frequency of channel openings and bursts. 6. Closed duration‐frequency histograms for GABA alone or with DZ or DMCM were best fitted by sums of at least six exponential functions. The three shortest closed duration time constants were unchanged by DZ or DMCM. The three longest closed duration time constants were altered by DZ and DMCM, consistent with alterations in opening frequency. 7. DZ increased and DMCM decreased steady‐state GABAA receptor current by increasing or decreasing channel opening frequency without altering mean channel open duration. We propose that DZ and DMCM alter GABAA receptor current by acting reciprocally to increase or decrease only, respectively, the apparent agonist association rate at the first of two proposed GABA binding steps without altering channel gating.(ABSTRACT TRUNCATED AT 400 WORDS)

Barbiturate regulation of kinetic properties of the GABAA receptor channel of mouse spinal neurones in culture.

1. Barbiturate regulation of the kinetic properties of gamma‐aminobutyric acidA (GABA) receptor channel chloride currents from somata of mouse spinal cord neurones were investigated using whole‐cell and excised outside‐out patch‐clamp recording techniques. 2. GABA (2 microM), GABA (2 microM) plus phenobarbitone (PhB) (500 microM) and GABA (2 microM) plus pentobarbitone (PB) (50 microM), applied by pressure ejection from blunt perfusion micropipettes, evoked inward chloride currents when neurones or patches were voltage clamped at ‐75 mV and the chloride equilibrium potential was 0 mV. GABA receptor channel currents were increased by PhB and PB. 3. Single GABA receptor channel currents were recorded with a main conductance state of 27 pS and a less frequent subconductance state of 16.5 pS. The conductances of the two states were unchanged by the barbiturates. 4. The main conductance state kinetics were analysed. GABA alone or with the barbiturates gated the channel open singly and in groups of openings. 5. The barbiturates increased GABA receptor channel mean open time and shifted frequency histograms of channel open times to longer times. 6. Three exponential functions were required to fit the frequency histograms of GABA receptor channel open times, suggesting that the channel has at least three open states (O1, O2, O3). The time constants for the exponential functions (0.9, 2.7 and 7.8 ms, respectively) were unchanged by the barbiturates. The increases in mean open times and the shifts of the open‐time frequency histograms by the barbiturates were due to a reduction in relative frequency of occurrence of the two short open states (O1 and O2) and to an increase in the relative frequency of occurrence of the longest open state (O3). 7. Frequency histograms of GABA receptor channel closed times were fitted with five exponential functions, suggesting that the channel has multiple closed states. None of the time constants nor areas of the exponential functions were significantly changed by the barbiturates. 8. For analysis, a burst was defined as openings surrounded by closures greater than a critical closed time, tc, of 5 ms. For GABA (2 microM), frequency histograms of GABA receptor channel bursts were fitted with three exponential functions, suggesting that the channel has three burst states (B1, B2, B3). The B1 burst state was probably a single opening to the O1 open state while the B2 and B3 burst states were probably composed of multiple openings to the O2 and O3 open states.(ABSTRACT TRUNCATED AT 400 WORDS)

Intraburst kinetic properties of the GABAA receptor main conductance state of mouse spinal cord neurones in culture.

1. The intraburst kinetic properties of the main conductance state of gamma‐aminobutyric acidA (GABAA) receptor channels in excised outside‐out patches obtained from somata of mouse spinal cord neurones in cell culture were investigated using the patch clamp single‐channel recording technique. 2. At 2 microM‐GABA, the burst duration distribution was fitted by four exponential functions with time constants of 0.5, 2.4, 8.3 and 31.8 ms. 3. At 0.5, 1 and 2 microM‐GABA, frequency distribution histograms of the number of apparent openings per burst were best fitted by three geometric functions with similar mean numbers (1.1, 1.9 and 3.6) of openings per burst. The proportion of bursts with a mean of 1.1 openings per burst decreased with increased GABA concentration while the proportion of bursts with means of 1.9 and 3.6 openings per burst increased with GABA concentration. 4. Analyses of GABA receptor channel intraburst kinetics were performed at all three GABA concentrations. The results were similar for all concentrations, but detailed results are presented only for 2 microM‐GABA. 5. The open time distribution for all intraburst openings was best fitted by three exponential functions with time constants of 0.6, 2.9 and 8.9 ms. 6. Intraburst open time and total open time distributions for bursts with one to five openings were fitted with two or three exponential functions or gamma distributions, respectively. The number of components, time constants and relative areas were similar for both distributions. 7. The distributions of open times for the nth opening within bursts of k openings were similar for bursts containing two to five openings. The distributions of open times for the nth opening of all bursts varied with position within the burst. The distributions shifted to longer openings as the opening number increased from one to five. 8. The distributions of all closings within all bursts or within bursts with two to five openings and of closings relative to position in all bursts could be fitted by two exponential functions with time constants of about 0.20 and 3.1 ms and relative proportions of 0.55 and 0.45 at all GABA concentrations. 9. The total closed time distributions for bursts containing two to four closings, however, were all best fitted with only a single gamma distribution with a time constant of 1.3 ms.(ABSTRACT TRUNCATED AT 400 WORDS)

Kinetic properties of the glycine receptor main‐ and sub‐conductance states of mouse spinal cord neurones in culture.

1. The kinetic properties of the two most frequent conductance states of glycine receptor channels from somata of mouse spinal cord neurones in cell culture were investigated using the outside‐out patch clamp technique. At low concentrations of glycine (0.5, 1 and 2 microM), single‐channel currents were recorded with two predominant amplitudes corresponding to a dominant or main‐conductance state of about 42 pS and a sub‐conductance state of about 27 pS. Both conductance states opened singly and in bursts of several openings. Total current evoked and single‐channel opening frequency increased as glycine concentration was increased from 0.5 to 2 microM. 2. For both conductance states mean open times were increased and open time frequency histograms were shifted to longer times as glycine concentration was increased from 0.5 to 2 microM. For both conductance states, three exponential components were required to fit best open time frequency distribution histograms at all glycine concentrations (0.5, 1 and 2 microM). The time constants of the exponential components for each conductance state were not significantly different across concentration, suggesting that the main‐ and sub‐conductance states of the channel each opened into at least three open states. For the main‐conductance state, the time constants were 1.09 +/‐ 0.09, 4.06 +/‐ 0.26 and 9.79 +/‐ 0.30 ms. For the sub‐conductance state, the time constants were 0.55 +/‐ 0.04, 2.64 +/‐ 0.35 and 8.57 +/‐ 1.08 ms. The increase in long open times with concentration was due primarily to a shift in relative frequency of occurrence of openings from the shortest to the two longest open states. 3. Closed time frequency distributions of closures between main‐conductance state openings, closures between sub‐conductance state openings and closures between both main‐ and sub‐conductance state openings were fitted with multiple exponential components, suggesting that the channel had several closed states. The two shortest time constants (0.16 +/‐ 0.01 and 1.26 +/‐ 0.13 ms) did not vary significantly with concentration (0.5‐2 microM) or method of analysis. The longer time constant varied with concentration. 4. Bursts were defined as groups of openings surrounded by closures greater than a critical closed time. For both conductances states, mean burst durations were increased and burst duration frequency histograms were shifted to longer times as glycine concentration was increased from 0.5 to 2 miroM. Burst duration frequency histograms contained four exponential components for the main‐conductance state and three exponential components for the sub‐conductance state.(ABSTRACT TRUNCATED AT 400 WORDS)

Pentobarbital and picrotoxin have reciprocal actions on single GABAA receptor channels

Pentobarbital (PB) and picrotoxin (PIC) bind to allosterically coupled sites on the GABAA receptor complex but have opposite effects on GABA receptor currents. PB, an anesthetic/anticonvulsant, enhances, and PIC, a convulsant, inhibits GABA receptor currents. PB and PIC also had opposite effects on single main conductance channel GABA receptor currents recorded from excised outside-out patches from mouse spinal neurons in culture. PB prolonged bursts of channel openings by increasing mean duration and number of intraburst openings. PIC shortened bursts by reducing mean duration and number of intraburst openings. The results demonstrate the reciprocal regulation of GABA receptor channels by PB and PIC and suggest that their allosterically coupled binding sites are coupled to the chloride channel in an opposite manner.

KINETIC PROPERTIES AND REGULATION OF GABAA RECEPTOR CHANNELS

Single-channel recordings of GABAA receptor single-channel currents have been obtained from mouse spinal cord neurons in cell culture. Detailed kinetic analysis of single-channel main-conductance level currents has allowed development of a preliminary kinetic scheme which describes the gating of the GABAA receptor channel. The essential features of this kinetic scheme are presented in scheme 1 (see above). In this scheme, the GABAA receptor channel is envisioned to exist in multiple open and closed states. Properties can be broken into three main categories. First, the receptor can exist in a closed and nondesensitized set of states. In the kinetic model it is envisioned that there is an unbound (C13), a singly bound (C12), and two doubly bound (C11 and C10) closed states. The singly bound and doubly bound closed states are thought to open to three open states (O1, O2, O3). However, each of the open states opens to two distal closed states whose kinetic properties are similar for all three open states (C4-C9). Only one desensitized state (D0) has been incorporated into the model. While this characterization of desensitization is certainly incomplete, it is an initial step toward including the desensitization process which is clearly evident in whole-cell and single-channel recordings. This kinetic scheme should be considered only an initial working model. A number of features appear to be correct. First, all analyses of open time frequency histograms for GABA- and GABA agonist-induced single-channel openings have demonstrated the presence of at least three distinct open time constants. Furthermore, the concentration-dependent change in the relative frequency of occurrence of the three open states suggests that the open states occur from singly and doubly bound forms of the receptor. Second, the presence of two brief closed states adjacent to the open states appears fairly secure. However, it should be noted that the kinetic analysis primarily suggests that each open state opens to two brief adjacent closed states in a concentration-independent manner. While we have indicated that these two closed states are distal to the three open states, the actual assignment of the location of these states is unclear. Another interpretation of these data is that there is only a single distal closed state and that the proximal, extraburst closed states have very brief durations that are similar to each other. What appears clear, however, is that the open states can close to either brief closed state.(ABSTRACT TRUNCATED AT 400 WORDS)

Kinetics of open channel block by penicillin of single GABAA receptor channels from mouse spinal cord neurones in culture.

1. Reduction by penicillin of single gamma‐aminobutyric acidA (GABAA) receptor currents from somata of mouse spinal cord neurones in culture was investigated using the excised outside‐out patch‐clamp recording technique. 2. GABA (2 microM) alone or with penicillin (100‐5000 microM) applied by pressure ejection from micropipettes evoked inward currents when patches were voltage‐clamped at ‐75 mV in symmetrical chloride solutions. Averaged GABA receptor currents were decreased in the presence of penicillin. 3. GABA receptor currents were recorded with at least two conductance states, a more frequent or main‐conductance state of about 27 pS and a less frequent sub‐conductance state of about 19‐20 pS. The conductances of the two states were unchanged in the presence of penicillin. The kinetic properties of the main‐conductance state were analysed and are summarized below. 4. Penicillin produced a concentration‐dependent reduction of GABA receptor open properties by reduction of average GABA receptor channel open duration and an increase in channel opening frequency. 5. Penicillin shifted frequency histograms of GABA receptor channel open durations to shorter durations in a concentration‐dependent manner. Three exponential functions were required to fit best the frequency histograms of open durations, suggesting that the channel had at least three open states. Penicillin produced a concentration‐dependent reduction in the time constants obtained from the open duration frequency histograms. 6. Frequency histograms of GABA receptor channel closed durations could be fitted with five to seven exponential functions, suggesting that the channel had multiple closed states. In the presence of increased concentration of penicillin, there was a reduction in the relative frequency of brief gaps and the appearance of new closed time constants. 7. With increased penicillin concentration, GABA receptor channel burst frequency was unchanged, burst durations were increased, the number of openings per burst was increased and the per cent time open within a burst was decreased. 8. The results suggested that penicillin produced simple open channel blockade of the GABA receptor channel. However, the experimental results also suggested that the association with and, perhaps, the dissociation of the blocker from its binding site were dependent upon the kinetic state of the open channel. Penicillin had faster association and slower dissociation rates when the channel was in unstable, brief open kinetic state than when the channel was in a more stable, longer open kinetic state. Possible models for penicillin reduction of single GABA receptor currents were simulated by computer and analysed.(ABSTRACT TRUNCATED AT 400 WORDS)