Paola Pedarzani

Differential Distribution of Three Ca2+-Activated K+ Channel Subunits, SK1, SK2, and SK3, in the Adult Rat Central Nervous System

Ca(2+)-activated, voltage-independent K(+) channels are present in most neurons and mediate the afterhyperpolarizations (AHPs) following action potentials. They present distinct physiological and pharmacological properties and play an important role in controlling neuronal firing frequency and spike frequency adaptation. We used in situ hybridization to characterize the distribution patterns of the three cloned SK channel subunits (SK1-3), the prime candidates likely to underlie Ca(2+)-dependent AHPs in the central nervous system. We found high levels of expression in regions presenting prominent AHP currents, such as, for example, neocortex and CA1-3 layers of the hippocampus (SK1 and SK2), reticularis thalami (SK1 and SK2), supraoptic nucleus (SK3), and inferior olivary nucleus (SK2 and SK3). Our results reveal the functional role of SK channels with defined subunit compositions in some neurons and open the way to the identification of the molecular determinants of AHP currents in many brain regions.

Specific enhancement of SK channel activity selectively potentiates the afterhyperpolarizing current I-AHP and modulates the firing properties of hippocampal pyramidal neurons

SK channels are Ca2+-activated K+ channels that underlie after hyperpolarizing (AHP) currents and contribute to the shaping of the firing patterns and regulation of Ca2+ influx in a variety of neurons. The elucidation of SK channel function has recently benefited from the discovery of SK channel enhancers, the prototype of which is 1-EBIO. 1-EBIO exerts profound effects on neuronal excitability but displays a low potency and limited selectivity. This study reports the effects of DCEBIO, an intermediate conductance Ca2+-activated K+ channel modulator, and the effects of the recently identified potent SK channel enhancer NS309 on recombinant SK2 channels, neuronal apamin-sensitive AHP currents, and the excitability of CA1 neurons. NS309 and DCEBIO increased the amplitude and duration of the apamin-sensitive afterhyperpolarizing current without affecting the slow afterhyperpolarizing current in contrast to 1-EBIO. The potentiation by DCEBIO and NS309 was reversed by SK channel blockers. In current clamp experiments, NS309 enhanced the medium afterhyperpolarization (but not the slow afterhyperpolarization sAHP) and profoundly affected excitability by facilitating spike frequency adaptation in a frequency-independent manner. The potent and specific effect of NS309 on the excitability of CA1 pyramidal neurons makes this compound an ideal tool to assess the role of SK channels as possible targets for the treatment of disorders linked to neuronal hyperexcitability.

Molecular determinants of Ca2+-dependent K+ channel function in rat dorsal vagal neurones

1 Using in situ hybridisation histochemistry in combination with patch‐clamp recordings and specific pharmacological tools, the molecular nature of the channels underlying Ca2+‐dependent K+ currents was determined in dorsal vagal neurones (DVNs) of rat brainstem slices. 2 In situ hybridisation analysis at cellular resolution revealed the presence of ‘big’‐conductance Ca2+‐ and voltage‐activated K+ (BK) channel α‐subunit mRNA, and of only one ‘small’‐conductance Ca2+‐activated K+ (SK) channel subunit transcript, SK3, at very high levels in DVNs. By contrast, SK1 and SK2 mRNAs were below the threshold limit of detection. 3 The SK channel‐mediated after‐hyperpolarising current (IAHP) was blocked by apamin with a half‐maximal inhibitory concentration of ∼2.2 nm. This is consistent with homomultimeric SK3 channels mediating IAHP in DVNs. IAHP was also blocked by scyllatoxin (20–30 nm) and curare (100–200 μm). 4 Application of apamin (100 nm) or scyllatoxin (20 nm) invariably caused a substantial increase to 146.1 ± 10.4 and 181.8 ± 12.9 % of control, respectively, in the spontaneous firing rate of DVNs. Action potential duration was not affected by these SK channel blockers. 5 The selective BK channel blocker iberiotoxin (50 nm) increased action potential duration by 22.5 ± 7.3 %, as did low concentrations of tetraethylammonium (0.5 mm; 99.3 ± 16.4 %) and the Ca2+ channel blocker Cd2+ (100 μm; 49.5 ± 20.9 %). BK channel blockade did not significantly affect the firing rate of DVNs. 6 These results allow us to establish a tight correlation between the properties of cloned and native BK and SK channels, and to achieve an understanding, at the molecular level, of their role in regulating the spontaneous firing frequency and in shaping single action potentials of central neurones.

Inhibitory Gating Modulation of Small Conductance Ca2+-Activated K+ Channels by the Synthetic Compound (R)-N-(Benzimidazol-2-yl)-1,2,3,4-tetrahydro-1-naphtylamine (NS8593) Reduces Afterhyperpolarizing Current in Hippocampal CA1 Neurons

SK channels are small conductance Ca2+-activated K+ channels important for the control of neuronal excitability, the fine tuning of firing patterns, and the regulation of synaptic mechanisms. The classic SK channel pharmacology has largely focused on the peptide apamin, which acts extracellularly by a pore-blocking mechanism. 1-Ethyl-2-benzimidazolinone (1-EBIO) and 6,7-dichloro-1H-indole-2,3-dione 3-oxime (NS309) have been identified as positive gating modulators that increase the apparent Ca2+ sensitivity of SK channels. In the present study, we describe inhibitory gating modulation as a novel principle for selective inhibition of SK channels. In wholecell patch-clamp experiments, the compound (R)-N-(benzimidazol-2-yl)-1,2,3,4-tetrahydro-1-naphtylamine (NS8593) reversibly inhibited recombinant SK3-mediated currents (human SK3 and rat SK3) with potencies around 100 nM. However, in contrast to known pore blockers, NS8593 did not inhibit 125I-apamin binding. Using excised patches, it was demonstrated that NS8593 decreased the Ca2+ sensitivity by shifting the activation curve for Ca2+ to the right, only slightly affecting the maximal Ca2+-activated SK current. NS8593 inhibited all the SK1-3 subtypes Ca2+-dependently (Kd = 0.42, 0.60, and 0.73 μM, respectively, at 0.5 μM Ca2+), whereas the compound did not affect the Ca2+-activated K+ channels of intermediate and large conductance (hIK and hBK channels, respectively). The site of action was accessible from both sides of the membrane, and the NS8593-mediated inhibition was prevented in the presence of a high concentration of the positive modulator NS309. NS8593 was further tested on mouse CA1 neurons in hippocampal slices and shown to inhibit the apaminand tubocurarine-sensitive SK-mediated afterhyperpolarizing current, at a concentration of 3 μM.

A protein phosphatase is involved in the cholinergic suppression of the Ca2+-activated K+ current sIAHP in hippocampal pyramidal neurons

The slow calcium-activated potassium current sI(AHP) underlies spike-frequency adaptation and has a substantial impact on the excitability of hippocampal CA1 pyramidal neurons. Among other neuromodulatory substances, sI(AHP) is modulated by acetylcholine acting via muscarinic receptors. The second-messenger systems mediating the suppression of sI(AHP) by muscarinic agonists are largely unknown. Both protein kinase C and A do not seem to be involved, whereas calcium calmodulin kinase II has been shown to take part in the muscarinic action on sI(AHP). We re-examined the mechanism of action of muscarinic agonists on sI(AHP) combining whole-cell recordings with the use of specific inhibitors or activators of putative constituents of the muscarinic pathway. Our results suggest that activation of muscarinic receptors reduces sI(AHP) in a G-protein-mediated and phospholipase C-independent manner. Furthermore, we obtained evidence for the involvement of the cGMP-cGK pathway and of a protein phosphatase in the cholinergic suppression of sI(AHP), whereas release of Ca(2+) from IP(3)-sensitive stores seems to be relevant neither for maintenance nor for modulation of sI(AHP).

Domain Analysis of the Calcium-activated Potassium Channel SK1 from Rat Brain FUNCTIONAL EXPRESSION AND TOXIN SENSITIVITY

Two small conductance, calcium-activated potassium channels (SK channels), SK2 and SK3, have been shown to contribute to the afterhyperpolarization (AHP) and to shape the firing behavior in neurons for example in the hippocampal formation, the dorsal vagal nucleus, the subthalamic nucleus, and the cerebellum. In heterologous expression systems, SK2 and SK3 currents are blocked by the bee venom toxin apamin, just as well as the corresponding neuronal AHP currents. However, the functional role and pharmacological profile of SK1 channels from rat brain (rSK1) is still largely unknown, as so far rSK1 homomeric channels could not be functionally expressed. We have performed a domain analysis to elucidate the pharmacological profile and the molecular determinants of rSK1 channel expression by using channel chimeras in combination with immunocytochemistry, immunoblot analysis, and electrophysiology. Our results reveal that the rSK1 subunit is synthesized in cells but does not form functional homomeric channels. Exchanging the carboxyl terminus of rSK1 for that of hSK1 or rSK2 is sufficient to rescue the functional expression of rSK1 channels. Additionally, transplantation of both amino and carboxyl termini of rSK1 onto hSK1 subunits, normally forming functional homomeric channel, hinders their functional expression, while hSK1 channels containing only the rSK1 carboxyl terminus are functional. These results suggest that the lack of functional expression of rSK1 channels is probably due to problems in their assembly and tetramerization but not in their calmodulin-dependent gating. Finally, we show that chimeric channels containing the core domain (S1–S6) of rSK1, unlike hSK1, are apamin-insensitive.

Chemical anoxia activates ATP-sensitive and blocks Ca2+-dependent K+ channels in rat dorsal vagal neurons in situ

The contribution of subclasses of K(+) channels to the response of mammalian neurons to anoxia is not yet clear. We investigated the role of ATP-sensitive (K(ATP)) and Ca(2+)-activated K(+) currents (small conductance, SK, big conductance, BK) in mediating the effects of chemical anoxia by cyanide, as determined by electrophysiological analysis and fluorometric Ca(2+) measurements in dorsal vagal neurons of rat brainstem slices. The cyanide-evoked persistent outward current was abolished by the K(ATP) channel blocker tolbutamide, but not changed by the SK and BK channel blockers apamin or tetraethylammonium. The K(+) channel blockers also revealed that ongoing activation of K(ATP) and SK channels counteracts a tonic, spike-related rise in intracellular Ca(2+) ([Ca(2+)](i)) under normoxic conditions, but did not modify the rise of [Ca(2+)](i) associated with the cyanide-induced outward current. Cyanide depressed the SK channel-mediated afterhyperpolarizing current without changing the depolarization-induced [Ca(2+)](i) transient, but did not affect spike duration that is determined by BK channels. The afterhyperpolarizing current and the concomitant [Ca(2+)](i) rise were abolished by Ca(2+)-free superfusate that changed neither the cyanide-induced outward current nor the associated [Ca(2+)](i) increase. Intracellular BAPTA for Ca(2+) chelation blocked the afterhyperpolarizing current and the accompanying [Ca(2+)](i) increase, but had no effect on the cyanide-induced outward current although the associated [Ca(2+)](i) increase was noticeably attenuated. Reproducing the cyanide-evoked [Ca(2+)](i) transient with the Ca(2+) pump blocker cyclopiazonic acid did not evoke an outward current. Our results show that anoxia mediates a persistent hyperpolarization due to activation of K(ATP) channels, blocks SK channels and has no effect on BK channels, and that the anoxic rise of [Ca(2+)](i) does not interfere with the activity of these K(+) channels.