An intermediate conductance calciumactivated potassium channel, hIK1, was cloned from human pancreas. The predicted amino acid sequence is related to, but distinct from, the small conductance calcium-activated potassium channel subfamily, which is Ϸ50% conserved. hIK1 mRNA was detected in peripheral tissues but not in brain. Expression of hIK1 in Xenopus oocytes gave rise to inwardly rectifying potassium currents, which were activated by submicromolar concentrations of intracellular calcium (K 0.5 ‫؍‬ 0.3 M). Although the K 0.5 for calcium was similar to that of small conductance calcium-activated potassium channels, the slope factor derived from the Hill equation was significantly reduced (1.7 vs. 3.5). Single-channel current amplitudes ref lected the macroscopic inward rectification and revealed a conductance level of 39 pS in the inward direction. hIK1 currents were reversibly blocked by charybdotoxin (K i ‫؍‬ 2.5 nM) and clotrimazole (K i ‫؍‬ 24.8 nM) but were minimally affected by apamin (100 nM), iberiotoxin (50 nM), or ketoconazole (10 M). These biophysical and pharmacological properties are consistent with native intermediate conductance calcium-activated potassium channels, including the erythrocyte Gardos channel.

Arachidonic acid (AA) is an important constituent of membrane phospholipids and can be liberated by activation of cellular phospholipases. AA modulates a variety of ion channels via diverse mechanisms, including both direct effects by AA itself and indirect actions through AA metabolites. Here, we report excitatory effects of AA on a cloned human inwardly rectifying K(+) channel, Kir2.3, which is highly expressed in the brain and heart and is critical in regulating cell excitability. AA potently and reversibly increased Kir2.3 current amplitudes in whole-cell and excised macro-patch recordings (maximal whole-cell response to AA was 258 +/- 21% of control, with an EC(50) value of 447 nM at -97 mV). This effect was apparently caused by an action of AA at an extracellular site and was not prevented by inhibitors of protein kinase C, free oxygen radicals, or AA metabolic pathways. Fatty acids that are not substrates for metabolism also potentiated Kir2.3 current. AA had no effect on the currents flowing through Kir2.1, Kir2.2, or Kir2.4 channels. Experiments with Kir2.1/2.3 chimeras suggested that, although AA may bind to both Kir2.1 and Kir2.3, the transmembrane and/or intracellular domains of Kir2.3 were essential for channel potentiation. These results argue for a direct mechanism of AA modulation of Kir2.3.

Voltage dependent sodium channels are widely recognized as valuable targets for the development of therapeutic interventions for neuroexcitatory disorders such as epilepsy and pain as well as cardiac arrhythmias. An ongoing challenge for sodium channel drug discovery is the ability to readily evaluate state dependent interactions, which are known to underlie inhibition by many clinically used local anesthetic, antiepileptic and antiarrhythmic sodium channel blockers. While patch-clamp electrophysiology is still considered the most effective way of measuring ion channel function and pharmacology, it does not have the throughput to be useful in early stages of drug discovery in which there is often a need to evaluate many thousands to hundreds of thousands of compounds. Fortunately over the past five years, there has been significant progress in developing much higher throughput electrophysiology platforms like the PatchXpress and IonWorks, which are now widely used in drug discovery. This review highlights the strengths and weaknesses of these two high throughput devices for use in sodium channel inhibitor drug discovery programs. Overall, the PatchXpress and IonWorks electrophysiology platforms have individual strengths that make them complementary to each other. Both platforms are capable of measuring state dependent modulation of sodium channels. IonWorks has the throughput to allow for effective screening of libraries of tens of thousands of compounds whereas the PatchXpress has more flexibility to provide quantitative voltage clamp, which is useful in structure activity evaluations for the hit-to-lead and lead optimization stages of sodium channel drug discovery.

Aromatic L-amino acid decarboxylase (AAAD) activity of rat retina increases when animals are placed in a lighted environment from the dark. The increase of activity can be inhibited by administering the selective dopamine D1 receptor agonist SKF 38393, but not the selective D2 agonist quinpirole, or apomorphine. Conversely, in the dark, enzyme activity can be enhanced by administering the selective D1 antagonist SCH 23390 or haloperidol, but not the selective D2 antagonist (-)-sulpiride. Furthermore, in animals exposed to room light for 3 h, the D1 agonist SKF 38393 reduced retinal AAAD activity, and this effect was prevented by prior administration of SCH 23390. In contrast, quinpirole had little or no effect when administered to animals in the light. Kinetic analysis indicated that the apparent Vmax for the enzyme increases with little change in the apparent Km for the substrate 3,4-dihydroxyphenylalanine or the cofactor pyridoxal-5'-phosphate. We suggest that dopamine released in the dark tonically occupies D1 receptors and suppresses AAAD activity. When the room light is turned on, D1 receptors are vacated and selective D1 agonists can either prevent the rise of AAAD or reverse light-enhanced AAAD activity.