Activity-dependent changes in neuronal excitability and synaptic strength are thought to underlie memory encoding. In hippocampal CA1 neurons, small conductance Ca 2ϩ -activated K ϩ (SK) channels contribute to the afterhyperpolarization, affecting neuronal excitability. In the present study, we examined the effect of apamin-sensitive SK channels on the induction of hippocampal synaptic plasticity in response to a range of stimulation frequencies. In addition, the role of apamin-sensitive SK channels on hippocampal-dependent memory encoding and retention was also tested. The results show that blocking SK channels with apamin increased the excitability of hippocampal neurons and facilitated the induction of synaptic plasticity by shifting the modification threshold to lower frequencies. This facilitation was NMDA receptor (NMDAR) dependent and appeared to be postsynaptic. Mice treated with apamin demonstrated accelerated hippocampal-dependent spatial and nonspatial memory encoding. They required fewer trials to learn the location of a hidden platform in the Morris water maze and less time to encode object memory in an object-recognition task compared with saline-treated mice. Apamin did not influence long-term retention of spatial or nonspatial memory. These data support a role for SK channels in the modulation of hippocampal synaptic plasticity and hippocampal-dependent memory encoding.

] >100 nM while having no effect on K s or n. Mutation of the single PKA consensus phosphorylation site at serine 334 to alanine (S334A) had no effect on the PKA-dependent activation during either two-electrode voltage-clamp or in excised inside-out patches. When expressed in HEK293 cells, ATP activated hIK1 in a Mg 2؉ -dependent fashion, being reversed by alkaline phosphatase. Neither PKI 5-24 nor CaMKII 281-309 or PKC 19 -31 affected the ATP-dependent activation. Northern blot analysis revealed hIK1 expression in the T84 colonic cell line. Endogenous hIK1 was activated by ATP in a Mg 2؉ -and PKI 5-24 -dependent fashion and was reversed by alkaline phosphatase, whereas CaMKII 281-309 and PKC 19 -31 had no effect on the ATP-dependent activation. The Ca 2؉ -dependent activation (K s and n) was unaffected by ATP. In conclusion, hIK1 is activated by a membrane delimited PKA when endogenously expressed. Although the oocyte expression system recapitulates this regulation, expression in HEK293 cells does not. The effect of PKA on hIK1 gating is Ca 2؉ -dependent and occurs via an increase in NP o without an effect on either Ca 2؉ affinity or apparent cooperativity.

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We evaluated the effects of clotrimazole and clofibrate on Ca(2+)- and adenosine 3',5'-cyclic monophosphate (cAMP)-mediated Cl- secretion in the colonic cell line, T84. We used 1-ethyl-2-benzimidazolinone (1-EBIO) to activate the Ca(2+)-dependent K+ channel (KCa) in these cells to induce a sustained Cl- secretory current (Isc). Clotrimazole potently inhibited the KCa-dependent Isc, with an inhibition constant (Ki) of 0.27 +/- 0.02 microM. Clofibrate also inhibited the 1-EBIO-induced Isc albeit with lower affinity (Ki = 6.5 +/- 1.2 microM). Clotrimazole (10 microM) inhibited the Isc response to the Ca(2+)-mediated agonist, carbachol, by 82%. Similarly, both clotrimazole and clofibrate inhibited cAMP-mediated Cl- secretion, with Ki values of 5.2 +/- 1.0 and 6.7 +/- 1.1 microM, respectively. We used nystatin to permeabilize the apical or basolateral membrane to determine the effects of clotrimazole and clofibrate on the basolateral K+ (IK) and apical Cl- (ICl) currents following stimulation by either 1-EBIO or forskolin. Both clotrimazole and clofibrate inhibited the 1-EBIO- and forskolin-induced IK without affecting ICl. We determined the effects of clotrimazole and clofibrate on KCa using 86Rb+ uptake studies into membrane vesicles. Both clotrimazole and clofibrate inhibited the 1-EBIO-induced 86Rb+ uptake, with Ki values of 0.31 +/- 0.08 and 10.8 +/- 5.5 microM, respectively. Similarly, clotrimazole inhibited the Ca(2+)-induced 86Rb+ uptake with a Ki of 0.51 +/- 0.15 microM. Charybdotoxin inhibited both the 1-EBIO- and Ca(2+)-induced 86Rb+ uptakes with similar affinities (Ki values of 0.57 +/- 0.07 and 0.47 +/- 0.08 nM, respectively), suggesting 1-EBIO and Ca2+ activate the same channel (KCa) in this assay. In excised, single-channel recordings both clotrimazole and clofibrate inhibited KCa, demonstrating a direct inhibition of the channel by these compounds. We demonstrate that clotrimazole blocks the intestinal KCa, thereby inhibiting Cl- secretion. These results suggest that clotrimazole may be useful as an antidiarrheal.

We previously characterized 1-ethyl-2-benzimidazolinone (1-EBIO), as well as the clinically useful benzoxazoles, chlorzoxazone (CZ), and zoxazolamine (ZOX), as pharmacological activators of the intermediate-conductance Ca(2+)-activated K(+) channel, hIK1. The mechanism of activation of hIK1, as well as the highly homologous small-conductance, Ca(2+)-dependent K(+) channel, rSK2, was determined following heterologous expression in Xenopus oocytes using two-electrode voltage clamp (TEVC) and excised, inside-out patch-clamp techniques. 1-EBIO, CZ, and ZOX activated both hIK1 and rSK2 in TEVC and excised inside-out patch-clamp experiments. In excised, inside-out patches, 1-EBIO and CZ induced a concentration-dependent activation of hIK1, with half-maximal (K(1/2)) values of 84 microM and 98 microM, respectively. Similarly, CZ activated rSK2 with a K(1/2) of 87 microM. In the absence of CZ, the Ca(2+)-dependent activation of hIK1 was best fit with a K(1/2) of 700 nM and a Hill coefficient (n) of 2.0. rSK2 was activated by Ca(2+) with a K(1/2) of 700 nM and an n of 2.5. Addition of CZ had no effect on either the K(1/2) or n for Ca(2+)-dependent activation of either hIK1 or rSK2. Rather, CZ increased channel activity at all Ca(2+) concentrations (V(max)). Event-duration analysis revealed hIK1 was minimally described by two open and three closed times. Activation by 1-EBIO had no effect on tau(o1), tau(o2), or tau(c1), whereas tau(c2) and tau(c3) were reduced from 9.0 and 92.6 ms to 5.0 and 44.1 ms, respectively. In conclusion, we define 1-EBIO, CZ, and ZOX as the first known activators of hIK1 and rSK2. Openers of IK and SK channels may be therapeutically beneficial in cystic fibrosis and vascular diseases.