F-box protein-32 down-regulates small-conductance calcium-activated potassium channel 2 in diabetic mouse atria.

Diabetes mellitus (DM) is an independent risk factor for atrial fibrillation, but the underlying ionic mechanism for this association remains unclear. We recently reported that expression of the small-conductance calcium-activated potassium channel 2 (SK2, encoded by KCCN2) in atria from diabetic mice is significantly down-regulated, resulting in reduced SK currents in atrial myocytes from these mice. We also reported that the level of SK2 mRNA expression is not reduced in DM atria but that the ubiquitin-proteasome system (UPS), a major mechanism of intracellular protein degradation, is activated in vascular smooth muscle cells in DM. This suggests a possible role of the UPS in reduced SK currents. To test this possibility, we examined the role of the UPS in atrial SK2 down-regulation in DM. We found that a muscle-specific E3 ligase, F-box protein 32 (FBXO-32, also called atrogin-1), was significantly up-regulated in diabetic mouse atria. Enhanced FBXO-32 expression in atrial cells significantly reduced SK2 protein expression, and siRNA-mediated FBXO-32 knockdown increased SK2 protein expression. Furthermore, co-transfection of SK2 with FBXO-32 complementary DNA in HEK293 cells significantly reduced SK2 expression, whereas co-transfection with atrogin-1ΔF complementary DNA (a nonfunctional FBXO-32 variant in which the F-box domain is deleted) did not have any effects on SK2. These results indicate that FBXO-32 contributes to SK2 down-regulation and that the F-box domain is essential for FBXO-32 function. In conclusion, DM-induced SK2 channel down-regulation appears to be due to an FBXO-32-dependent increase in UPS-mediated SK2 protein degradation.

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The Ca(V)3.3 calcium channel is the major sleep spindle pacemaker in thalamus.

Low-threshold (T-type) Ca(2+) channels encoded by the Ca(V)3 genes endow neurons with oscillatory properties that underlie slow waves characteristic of the non-rapid eye movement (NREM) sleep EEG. Three Ca(V)3 channel subtypes are expressed in the thalamocortical (TC) system, but their respective roles for the sleep EEG are unclear. Ca(V)3.3 protein is expressed abundantly in the nucleus reticularis thalami (nRt), an essential oscillatory burst generator. We report the characterization of a transgenic Ca(V)3.3(-/-) mouse line and demonstrate that Ca(V)3.3 channels are indispensable for nRt function and for sleep spindles, a hallmark of natural sleep. The absence of Ca(V)3.3 channels prevented oscillatory bursting in the low-frequency (4-10 Hz) range in nRt cells but spared tonic discharge. In contrast, adjacent TC neurons expressing Ca(V)3.1 channels retained low-threshold bursts. Nevertheless, the generation of synchronized thalamic network oscillations underlying sleep-spindle waves was weakened markedly because of the reduced inhibition of TC neurons via nRt cells. T currents in Ca(V)3.3(-/-) mice were <30% compared with those in WT mice, and the remaining current, carried by Ca(V)3.2 channels, generated dendritic [Ca(2+)](i) signals insufficient to provoke oscillatory bursting that arises from interplay with Ca(2+)-dependent small conductance-type 2 K(+) channels. Finally, naturally sleeping Ca(V)3.3(-/-) mice showed a selective reduction in the power density of the σ frequency band (10-12 Hz) at transitions from NREM to REM sleep, with other EEG waves remaining unaltered. Together, these data identify a central role for Ca(V)3.3 channels in the rhythmogenic properties of the sleep-spindle generator and provide a molecular target to elucidate the roles of sleep spindles for brain function and development.

A novel isoform of SK2 assembles with other SK subunits in mouse brain.

The SK2 subtype of small conductance Ca2+-activated K+ channels is widely distributed throughout the central nervous system and modulates neuronal excitability by contributing to the afterhyperpolarization that follows an action potential. Western blots of brain membrane proteins prepared from wild type and SK2-null mice reveal two isoforms of SK2, a 49-kDa band corresponding to the previously reported SK2 protein (SK2-S) and a novel 78-kDa form. Complementary DNA clones from brain and Western blots probed with an antibody specific for the longer form, SK2-L, identified the larger molecular weight isoform as an N-terminally extended SK2 protein. The N-terminal extension of SK2-L is cysteine-rich and mediates disulfide bond formation between SK2-L subunits or with heterologous proteins. Immunohistochemistry revealed that in brain SK2-L and SK2-S are expressed in similar but not identical patterns. Heterologous expression of SK2-L results in functional homomeric channels with Ca2+ sensitivity similar to that of SK2-S, consistent with their shared core and intracellular C-terminal domains. In contrast to the diffuse, uniform surface distribution of SK2-S, SK2-L channels cluster into sharply defined, distinct puncta suggesting that the extended cysteine-rich N-terminal domain mediates this process. Immunoprecipitations from transfected cells and mouse brain demonstrate that SK2-L co-assembles with the other SK subunits. Taken together, the results show that the SK2 gene encodes two subunit proteins and suggest that native SK2-L subunits may preferentially partition into heteromeric channel complexes with other SK subunits.