Voltage-activated potassium channels in mammalian neurons and their block by novel pharmacological agents.

1. Electrophysiological studies have shown that a number of different types of potassium (K) channel currents exist in mammalian neurons. Among them are the voltage-gated K channel-currents which have been classified as fast-inactivating A-type currents (KA) and slowly inactivating delayed-rectifier type currents (KDR). 2. Two major molecular superfamilies of K channel have been identified; the KIR superfamily and the Shaker-related superfamily with a number of different pore-forming alpha-subunits in each superfamily. 3. Within the Shaker-related superfamily are the KV family, comprising of at least 18 different alpha-subunits that almost certainly underlie classically defined KA and KDR currents. However, the relationship between each of these cloned alpha-subunits and native voltage-gated K currents remains, for the most part, to be established. 4. Classical pharmacological blockers of voltage-gated K channels such as tetraethylammonium ions (TEA), 4-aminopyridine (4-AP), and certain toxins lack selectivity between different native channel currents and between different cloned K channel currents. 5. A number of other agents block neuronal voltage-gated K channels. All of these compounds are used primarily for other actions they possess. They include organic calcium (Ca) channel blockers, divalent and trivalent metal ions and certain calcium signalling agents such as caffeine. 6. A number of clinically active tricyclic compounds such as imipramine, amitriptyline, and chlorpromazine are also potent inhibitors of neuronal voltage-gated K channels. These compounds are weak bases and it appears that their uncharged form is required for activity. These compounds may provide a useful starting point for the rational design of novel selective K channel blocking agents.

Pharmacological and physiological characterization of murine homomeric beta3 GABA(A) receptors.

Gamma-Aminobutyric acid (GABA[A]) receptor beta3 subunits were expressed in Xenopus laevis oocytes and studied using two-electrode voltage clamp. Injected oocytes exhibited an increased resting membrane conductance and more depolarized membrane potentials compared to uninjected control cells. Oocytes expressing beta3 subunits were insensitive to GABA and muscimol, but pentobarbitone increased the membrane conductance in a concentration-dependent manner. The membrane current response to pentobarbitone reversed at the Cl- equilibrium potential and at relatively high concentrations (> 500 microM), a rebound Cl- current was induced following the removal of pentobarbitone. In transfected human embryonic kidney (HEK) cells, the rebound current amplitude was reduced by desensitizing the beta3 receptor with increased durations of ligand application. Both picrotoxin (0.5 nM to 10 microM) and Zn2+ (10 nM to 100 microM) reduced the resting membrane conductance for beta3 cDNA-injected oocytes. These oocytes were insensitive to flurazepam (5 microM) and alphaxalone (10 microM), but responded with increased membrane conductance to propofol (10 microM) and pregnanolone (50 nM to 5 microM). The antagonists, bicuculline (10 microM) and strychnine (50 nM to 100 microM), also induced conductance increases in a concentration dependent manner; however, glycine (1 mM) was inactive. It was concluded that beta3 subunits form spontaneously opening ion channels that can be up-regulated by some allosteric modulators, principally by pentobarbitone and propofol and, surprisingly, by bicuculline and strychnine, whilst picrotoxin and Zn2+ acted as antagonists. Computer modelling of some kinetic schemes was used to describe the rebound current observed in transfected HEK cells. This indicated that pentobarbitone, after modulation of the conductance, is potentially capable of further binding to the beta3 receptor complex 'driving' the receptor into one or more desensitized states. This phenomenon may be of some importance for native neuronal GABA(A) receptors, where pentobarbitone can also evoke rebound current activation.

Identification of a Zn2+ binding site on the murine GABAA receptor complex: dependence on the second transmembrane domain of beta subunits.

1. Whole-cell currents were recorded from Xenopus laevis oocytes expressing wild-type and mutant recombinant GABAA receptors to locate a binding site for Zn2+ ions in the beta 3 subunit. 2. The Cl(-)-selective current, spontaneously gated by beta 3 subunit homomers, was enhanced by pentobarbitone and inhibited by picrotoxinin. The potencies of these agents were minimally affected by mutating histidine (H) 292 to alanine (A) in the second transmembrane domain (TM2). 3. Zn2+ inhibited the beta 3 subunit-gated conductance (IC50, 0.31 microM); the inhibition was voltage insensitive. The H292A mutation in beta 3 subunits caused a 1000-fold reduction in Zn2+ potency (IC50, 307 microM). 4. GABA-activated responses recorded from heteromeric alpha 1 beta 3 GABAA receptors were also inhibited by Zn2+ (IC50, 0.11 microM). This inhibition was reduced by mutating H292A in the beta 3 subunit (IC50, 22.8 microM). 5. H292 in TM2 of the beta 3 subunit is an important determinant of a Zn2+ binding site on the GABAA receptor. Its location in the presumed ion channel lining suggests that Zn2+ can penetrate into an anion-selective channel and that the ionic selectivity filter and channel gate are located beyond H292.

Subcellular localization of gamma-aminobutyric acid type A receptors is determined by receptor beta subunits.

gamma-aminobutyric acid type A (GABAA) receptors are the major sites of fast synaptic inhibition in the brain. They are constructed from four subunit classes with multiple members: alpha (1-6), beta (1-4), gamma (1-4), and delta (1). The contribution of subunit diversity in determining receptor subcellular targeting was examined in polarized Madin-Darby canine kidney (MDCK) cells. Significant detection of cell surface homomeric receptor expression by a combination of both immunological and electrophysiological methodologies was only found for the beta 3 subunit. Expression of alpha/beta binary combinations resulted in a nonpolarized distribution for alpha 1 beta 1 complexes, but specific basolateral targeting of both alpha 1 beta 2 and alpha 1 beta 3 complexes. The polarized distribution of these alpha/beta complexes was unaffected by the presence of the gamma 2S subunit. Interestingly, delivery of receptors containing the beta 3 subunit to the basolateral domain occurs via the apical surface. These results show that beta subunits can selectively target GABAA receptors to distinct cellular locations. Changes in the spatial and temporal expression of beta-subunit isoforms may therefore provide a mechanism for relocating GABAA receptor function between distinct neuronal domains. Given the critical role of these receptors in mediating synaptic inhibition, the contribution of different beta subunits in GABAA receptor function, may have implications in neuronal development and for receptor localization/clustering.

Potent block of potassium currents in rat isolated sympathetic neurones by the uncharged form of amitriptyline and related tricyclic compounds.

1. The block of K+ currents by amitriptyline and the related tricyclic compounds cyproheptadine and dizocilpine was studied in dissociated rat sympathetic neurones by whole-cell voltage-clamp recording. 2. Cyproheptadine (30 microM) inhibited the delayed-rectifier current (Kv) by 92% and the transient current (KA) by 43%. For inhibition of Kv, cyproheptaidine had a KD of 2.2 microM. Dizocilpine (30 microM) inhibited Kv by 26% and KA by 22%. The stereoisomers of dizocilpine were equally potent at blocking Kv and KA. 3. Amitriptyline, a weak base, was significantly more effective in blocking Kv at pH 9.4 (KD = 0.46 microM) where the ratio of charged to uncharged drug was 50:50 compared with pH 7.4 (KD = 11.9 microM) where the ratio was 99:1. 4. N-methylamitriptyline (10 microM), the permanently charged analogue of amitriptyline, inhibited Kv by only 2% whereas in the same cells amitriptyline (10 microM) inhibited Kv by 36%. 5. Neither amitriptyline nor N-methylamitriptyline had a detectable effect on Kv when added to the intracellular solution. 6. It is concluded that the uncharged form of amitriptyline is approximately one hundred times more potent in blocking Kv than the charged form. However, this does not seem to be due to uncharged amitriptyline having better access to an intracellular binding site.

Block of potassium currents in rat isolated sympathetic neurones by tricyclic antidepressants and structurally related compounds.

1. The block of K+ currents by the tricyclic antidepressants (TCAs), imipramine and amitriptyline and three structurally related compounds, chlorpromazine, tacrine and carbamazepine was investigated in rat isolated sympathetic neurones by whole-cell voltage-clamp recording. 2. At a concentration of 10 microM, imipramine, amitriptyline and chlorpromazine all blocked the delayed rectifier K+ current (IKv) by about the same extent, 54%, 47% and 53%. Tacrine was less effective (10%) while carbamazepine was ineffective at all concentrations tested. 3. The degree of block by the four effective compounds was relatively independent of the size of the voltage-step. Neither the activation nor the inactivation rates of IKv were altered by the blocking drugs. 4. Concentration-response relationships for imipramine and tacrine showed that imipramine was about 7 fold more potent than tacrine but that the maximum inhibition and the Hill slope were the same for both compounds. 5. Amitriptyline, chlorpromazine and imipramine (at 10 microM) were 2-3 fold more potent at inhibiting the sustained K+ current (mostly IKv) than the transient K+ current (mostly IA). Tacrine, however, was equally effective in blocking both components.