Khaled M. Houamed

MODULATION OF RECOMBINANT AND NATIVE NEURONAL SK CHANNELS BY THE NEUROPROTECTIVE DRUG RILUZOLE

Small conductance, Ca(2+)-activated K(+) channels (SK channels) regulate neuronal excitability. We used patch clamp to study the actions of the neuroprotective drug riluzole on recombinant SK2 channels expressed in HEK293 cells and native SK channels underlying the afterhyperpolarization current (I(AHP)) in cultured hippocampal neurons. External riluzole activated whole-cell SK2 channel currents in HEK293 cells dialyzed with a Ca(2+)-free intracellular solution. When applied to the intracellular aspect of the membrane of giant inside-out patches, riluzole enhanced the membrane current activated by 100 nM Ca(2+) in a reversible and concentration-dependent manner; 30 microM riluzole applied to the intracellular aspect of the patches sensitized the channels to activation by Ca(2+), resulting in a leftward shift of the Ca(2+) activation curve. Riluzole also enhanced the I(AHP) and reduced the spontaneous action potential frequency in chemically stimulated neurons. Modulation of SK channel activity by riluzole may contribute to its cellular, behavioral, and clinical effects.

Pharmacological Properties and Functional Role of Kslow Current in Mouse Pancreatic β-Cells SK Channels Contribute to Kslow Tail Current and Modulate Insulin Secretion

The pharmacological properties of slow Ca2+-activated K+ current (Kslow) were investigated in mouse pancreatic β-cells and islets to understand how Kslow contributes to the control of islet bursting, [Ca2+]i oscillations, and insulin secretion. Kslow was insensitive to apamin or the KATP channel inhibitor tolbutamide, but UCL 1684, a potent and selective nonpeptide SK channel blocker reduced the amplitude of Kslow tail current in voltage-clamped mouse β-cells. Kslow was also selectively and reversibly inhibited by the class III antiarrythmic agent azimilide (AZ). In isolated β-cells or islets, pharmacologic inhibition of Kslow by UCL 1684 or AZ depolarized β-cell silent phase potential, increased action potential firing, raised [Ca2+]i, and enhanced glucose-dependent insulin secretion. AZ inhibition of Kslow also supported mediation by SK, rather than cardiac-like slow delayed rectifier channels since bath application of AZ to HEK 293 cells expressing SK3 cDNA reduced SK current. Further, AZ-sensitive Kslow current was extant in β-cells from KCNQ1 or KCNE1 null mice lacking cardiac slow delayed rectifier currents. These results strongly support a functional role for SK channel-mediated Kslow current in β-cells, and suggest that drugs that target SK channels may represent a new approach for increasing glucose-dependent insulin secretion. The apamin insensitivity of β-cell SK current suggests that β-cells express a unique SK splice variant or a novel heteromultimer consisting of different SK subunits.

Block of rat brain recombinant SK channels by tricyclic antidepressants and related compounds

SK channels are small conductance, Ca(2+)-activated K(+) channels that underlie neuronal slow afterhyperpolarization and mediate spike frequency adaptation. Using the patch clamp technique, we tested the effects of eight clinically relevant psychoactive compounds structurally related to the tricyclic antidepressants, on SK2 subtype channels cloned from rat brain and functionally expressed in the human embryonic kidney cell line, HEK293. Amitriptyline, carbamazepine, chlorpromazine, cyproheptadine, imipramine, tacrine and trifluperazine blocked SK2 channel currents with micromolar affinity. The block was reversible and concentration-dependent. The potency differed according to chemical structure. In contrast, the cognitive enhancer linopirdine was ineffective at blocking these channels. Our results point to a distinct pharmacological profile for SK channels.

Patch-clamp analysis of anesthetic interactions with recombinant SK2 subtype neuronal calcium-activated potassium channels

Small conductance calcium-activated potassium channels (SK) mediate spike frequency adaptation and underlie the slow afterhyperpolarization in central neurons. We tested the actions of several anesthetics on the SK2 subtype of recombinant SK channels, cloned from rat brain and functionally expressed in a mammalian cell line. Butanol, ethanol, ketamine, lidocaine, and methohexital blocked recombinant SK2 channel currents, measured in the whole-cell patch clamp recording mode. The block was reversible, dose-dependent, and of variable efficacy. The inhaled anesthetics chloroform, desflurane, enflurane, halothane, isoflurane, and sevoflurane produced little or no block when applied at 1 minimum alveolar anesthetic concentration; varying degrees of modulation were observed at very large concentrations (10 minimum alveolar concentration). The extent of block by inhaled anesthetics did not appear to depend on concentration or membrane voltage. Implications: We describe differential effects of anesthetics on cloned small conductance calcium-activated potassium channels from brain that may play a role in generating the effects or side effects of anesthetics.

Preserved BK Channel Function in Vasospastic Myocytes from a Dog Model of Subarachnoid Hemorrhage

Cerebral vasospasm after subarachnoid hemorrhage (SAH) is due to contraction of smooth muscle cells in the cerebral arteries. The mechanism of this contraction, however, is not well understood. Smooth muscle contraction is regulated in part by membrane potential, which is determined by K+ conductance in smooth muscle. Voltage-gated (Kv) and large-conductance, Ca2+-activated K+ (BK) channels dominate arterial smooth muscle K+ conductance. Vasospastic smooth muscle cells are depolarized relative to normal cells, but whether this is due to altered Kv or BK channel function has not been determined. This study determined if BK channels are altered during vasospasm after SAH in dogs. We first characterized BK channels in basilar-artery smooth muscle using whole-cell patch clamping and single-channel recordings. Next, we compared BK channel function between normal and vasospastic cells. There were no significant differences between normal and vasospastic cells in BK current density, kinetics, Ca2+ and voltage sensitivity, single-channel conductance or apparent Ca2+ affinity. Basilar-artery myocytes had no, small- or intermediate-conductance, Ca2+-activated K+ channels. The lack of difference in BK channels between vasospastic and control cells suggests alteration(s) in other K+ channels or other ionic conductances may underlie the membrane depolarization and vasoconstriction observed during vasospasm after SAH.

Activation of recombinant human SK4 channels by metal cations

The effects of metal cations on the activation of recombinant human SK4 (also known as hIK1 or hKCa4) channels, expressed in HEK 293 cells, were tested using patch clamp recording. Of the nine metals tested, cobalt, iron, magnesium, and zinc did not activate the SK4 channels when applied, at concentrations up to 100 μM, to the inside of SK4 channel‐expressing membrane patches. Barium, cadmium, calcium, lead, and strontium activated SK4 channels in a concentration‐dependent manner. The rank order of potency was at Ca2+>Pb2+>Cd2+>Sr2+>Ba2+.

Temporal profile of potassium channel dysfunction in cerebrovascular smooth muscle after experimental subarachnoid haemorrhage

The pathogenesis of cerebral vasospasm after subarachnoid haemorrhage (SAH) involves sustained contraction of arterial smooth muscle cells that is maximal 6-8 days after SAH. We reported that function of voltage-gated K+ (KV) channels was significantly decreased during vasospasm 7 days after SAH in dogs. Since arterial constriction is regulated by membrane potential that in turn is determined predominately by K+ conductance, the compromised K+ channel dysfunction may cause vasospasm. Additional support for this hypothesis would be demonstration that K+ channel dysfunction is temporally coincident with vasospasm. To test this hypothesis, SAH was created using the double haemorrhage model in dogs and smooth muscle cells from the basilar artery, which develops vasospasm, were isolated 4 days (early vasospasm), 7 days (during vasospasm) and 21 days (after vasospasm) after SAH and studied using patch-clamp electrophysiology. We investigated the two main K+ channels (KV and large-conductance voltage/Ca2+-activated (KCa) channels). Electrophysiologic function of KCa channels was preserved at all times after SAH. In contrast, function of KV channels was significantly decreased at all times after SAH. The decrease in cell size and degree of KV channel dysfunction was maximal 7 days after SAH. The results suggest that KV channel dysfunction either only partially contributes to vasospasm after SAH or that compensatory mechanisms develop that lead to resolution of vasospasm before KV channels recover their function.

Giant excised patch recordings of recombinant ion channel currents expressed in mammalian cells

The giant excised patch variant of patch clamp recording combines microsecond time resolution of macroscopic currents with rapid exchange of the experimental solutions at the intracellular membrane surface. This technique has been applied to a limited number of cell types, including Xenopus oocytes, muscle cells, and photoreceptors. We have applied this technique to recording recombinant ion channel currents expressed in membrane patches excised from HEK293 cell lines. Giant inside-out patch recordings of Na(+) channels and SK(Ca) type calcium-activated potassium channels show high temporal resolution and excellent signal to noise characteristics. This technique will facilitate the study of recombinant ion channels expressed in mammalian cells.