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The IK channel, a potassium ion channel regulated by calcium ions and voltages in a bidirectional manner, has been implicated in a range of diseases. However, there are currently few compounds available that can target the IK channel with high potency and specificity. Hainantoxin-I (HNTX-I) is the first peptide activator of IK channel discovered so far, but its activity is not ideal, and the underlying mechanism interaction between HNTX-I toxin and IK channel remains unclear. Thus, our study aimed to enhance the potency of IK channel activating peptides derived from HNTX-I and elucidate the molecular mechanism underlying the interaction between HNTX-I and the IK channel. By employing virtual alanine scanning mutagenesis, we generated 11 HNTX-I mutants using site-directed mutagenesis to pinpoint specific residues crucial for the HNTX-I and IK channel interaction. Subsequently, we identified key residues on the IK channel that are involved in the interaction with HNTX-I. Additionally, molecular docking was employed to guide the molecular engineering process and clarify the binding interface between HNTX-I and the IK channel. Our results demonstrate that HNTX-I primarily acts on the IK channel via the N-terminal amino acid, and its interaction with the IK channel is mediated by electrostatic and hydrophobic interactions, specifically the amino acid residues at positions 1, 3, 5, and 7 on HNTX-I. This study provides valuable insights into the peptide toxins that may serve as potential templates for the development of activators with enhanced potency and selectivity for the IK channel.

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Cooperative gating of localized ion channels ranges from fine-tuning excitation-contraction coupling in muscle cells to controlling pace-making activity in the heart. Membrane deformation resulting from muscle contraction activates stretch-activated (SA) cation channels. The subsequent Ca2+ influx activates spatially localized Ca2+-sensitive K+ channels to fine-tune spontaneous muscle contraction. To characterize endogenously expressed intermediate conductance Ca2+-activated potassium (IK) channels and assess the functional relevance of the extracellular Ca2+ source leading to IK channel activity, we performed patch-clamp techniques on cricket oviduct myocytes and recorded single-channel data. In this study, we first investigated the identification of IK channels that could be distinguished from endogenously expressed large-conductance Ca2+-activated potassium (BK) channels by adding extracellular Ba2+. The single-channel conductance of the IK channel was 62 pS, and its activity increased with increasing intracellular Ca2+ concentration but was not voltage-dependent. These results indicated that IK channels are endogenously expressed in cricket oviduct myocytes. Second, the Ca2+ influx pathway that activates the IK channel was investigated. The absence of extracellular Ca2+ or the presence of Gd3+ abolished the activity of IK channels. Finally, we investigated the proximity between SA and IK channels. The removal of extracellular Ca2+, administration of Ca2+ to the microscopic region in a pipette, and application of membrane stretching stimulation increased SA channel activity, followed by IK channel activity. Membrane stretch-induced SA and IK channel activity were positively correlated. However, the emergence of IK channel activity and its increase in response to membrane mechanical stretch was not observed without Ca2+ in the pipette. These results strongly suggest that IK channels are endogenously expressed in cricket oviduct myocytes and that IK channel activity is regulated by neighboring SA channel activity. In conclusion, functional coupling between SA and IK channels may underlie the molecular basis of spontaneous rhythmic contractions.

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Ion channels activated around the subthreshold membrane potential determine the likelihood of neuronal firing in response to synaptic inputs, a process described as intrinsic neuronal excitability. Long-term plasticity of chemical synaptic transmission is traditionally considered the main cellular mechanism of information storage in the brain; however, voltage- and calcium-activated channels modulating the inputs or outputs of neurons are also subjects of plastic changes and play a major role in learning and memory formation. Gamma oscillations are associated with numerous higher cognitive functions such as learning and memory, but our knowledge of their dependence on intrinsic plasticity is by far limited. Here we investigated the roles of potassium and calcium channels activated at near subthreshold membrane potentials in cholinergically induced persistent gamma oscillations measured in the CA3 area of rat hippocampal slices. Among potassium channels, which are responsible for the afterhyperpolarization in CA3 pyramidal cells, we found that blockers of SK (KCa2) and KV7.2/7.3 (KCNQ2/3), but not the BK (KCa1.1) and IK (KCa3.1) channels, increased the power of gamma oscillations. On the contrary, activators of these channels had an attenuating effect without affecting the frequency. Pharmacological blockade of the low voltage-activated T-type calcium channels (CaV3.1-3.3) reduced gamma power and increased the oscillation peak frequency. Enhancement of these channels also inhibited the peak power without altering the frequency of the oscillations. The presented data suggest that voltage- and calcium-activated ion channels involved in intrinsic excitability strongly regulate the power of hippocampal gamma oscillations. Targeting these channels could represent a valuable pharmacological strategy against cognitive impairment.

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The KCa 3.1 channel (KCNN4) is an important modulator of microglia responses in rodents, but no information exists on functional expression on microglia from human adults. We isolated and cultured microglia (max 1% astrocytes, no neurons or oligodendrocytes) from neocortex surgically removed from epilepsy patients and employed electrophysiological whole-cell measurements and selective pharmacological tools to elucidate functional expression of KCa 3.1. The channel expression was demonstrated as a significant increase in the voltage-independent current by NS309, a KCa 3.1/KCa 2 activator, followed by full inhibition upon co-application with NS6180, a highly selective KCa 3.1 inhibitor. A major fraction (79%) of unstimulated human microglia expressed KCa 3.1, and the difference in current between full activation and inhibition (ΔKCa 3.1) was estimated at 292 ± 48 pA at -40 mV (n = 75), which equals at least 585 channels per cell. Serial KCa 3.1 activation/inhibition significantly hyperpolarized/depolarized the membrane potential. The isolated human microglia were potently activated by lipopolysaccharide (LPS) shown as a prominent increase in TNF-α production. However, incubation with LPS neither changed the KCa 3.1 current nor the fraction of KCa 3.1 expressing cells. In contrast, the anti-inflammatory cytokine IL-4 slightly increased the KCa 3.1 current per cell, but as the membrane area also increased, there was no significant change in channel density. A large fraction of the microglia also expressed a voltage-dependent current sensitive to the KCa 1.1 modulators NS1619 and Paxilline and an inward-rectifying current with the characteristics of a Kir channel. The high functional expression of KCa 3.1 in microglia from epilepsy patients accentuates the need for further investigations of its role in neuropathological processes. GLIA 2016;64:2065-2078.

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This short review discusses pharmacological modulation of the opening/closing properties (gating) of small- and intermediate-conductance Ca(2+)-activated K(+) channels (KCa2 and KCa3.1) with special focus on mechanisms-of-action, selectivity, binding sites, and therapeutic potentials. Despite KCa channel gating-modulation being a relatively novel field in drug discovery, efforts in this area have already revealed a surprising plethora of pharmacological sites-of-actions and channel subtype selectivity exerted by different chemical classes. The currently published positive modulators show that such molecules are potentially useful for the treatment of various neurodegenerative disorders such as ataxia, alcohol dependence, and epilepsy as well as hypertension. The negative KCa2 modulators are very effective agents for atrial fibrillation. The prediction is that further unraveling of the molecular details of gating pharmacology will allow for the design of even more potent and subtype selective KCa modulators entering into drug development for these indications.

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