Large-conductance Ca 2ϩ -activated K ϩ channels (BK, also called Maxi-K or Slo channels) are widespread in the vertebrate nervous system, but their functional roles in synaptic transmission in the mammalian brain are largely unknown. By combining electrophysiology and immunogold cytochemistry, we demonstrate the existence of functional BK channels in presynaptic terminals in the hippocampus and compare their functional roles in somata and terminals of CA3 pyramidal cells. Doublelabeling immunogold analysis with BK channel and glutamate receptor antibodies indicated that BK channels are targeted to the presynaptic membrane facing the synaptic cleft in terminals of Schaffer collaterals in stratum radiatum. Whole-cell, intracellular, and field-potential recordings from CA1 pyramidal cells showed that the presynaptic BK channels are activated by calcium influx and can contribute to repolarization of the presynaptic action potential (AP) and negative feedback control of Ca 2ϩ influx and transmitter release. This was observed in the presence of 4-aminopyridine (4-AP, 40-100 M), which broadened the presynaptic compound action potential. In contrast, the presynaptic BK channels did not contribute significantly to regulation of action potentials or transmitter release under basal experimental conditions, i.e., without 4-AP, even at high stimulation frequencies. This is unlike the situation in the parent cell bodies (CA3 pyramidal cells), where BK channels contribute strongly to action potential repolarization. These results indicate that the functional role of BK channels depends on their subcellular localization.
Small-conductance Ca 2ϩ -activated K ϩ (SK) channels are important for excitability control and afterhyperpolarizations in vertebrate neurons and have been implicated in regulation of the functional state of the forebrain. We have examined the distribution, functional expression, and subunit composition of SK channels in rat brain. Immunoprecipitation detected solely homotetrameric SK2 and SK3 channels in native tissue and their constitutive association with calmodulin. Immunohistochemistry revealed a restricted distribution of SK1 and SK2 protein with highest densities in subregions of the hippocampus and neocortex. In contrast, SK3 protein was distributed more diffusely in these brain regions and predominantly expressed in phylogenetically older brain regions. Whole-cell recording showed a sharp segregation of apamin-sensitive SK current within the hippocampal formation, in agreement with the SK2 distribution, suggesting that SK2 homotetramers underlie the apamin-sensitive medium afterhyperpolarizations in rat hippocampus.
Molecular recognition of single NTA‐His6 pairs was detected with the atomic force microscope. The force required to dissociate a NTA‐His6 bond (unbinding force) was logarithmically dependent on the loading rate of the experiment and varied from 150 pN at 4.5 nN/s to 194 pN at 70 nN/s. Blocking experiments with imidazol clearly demonstrated the specificity of the detected recognition events. The lifetime of the NTA‐His6 bond decreased with increasing loading force, from 17 ms at 150 pN to 2.5 ms at 194 pN. Extrapolation with a Boltzmann ansatz lead to 15 s lifetime at zero force. The bond length of the NTA‐His6 binding pocket was estimated to be in the range of 0.2 nm. Since the interaction force of NTA‐His6 is considerably higher than the typical binding strength of other receptor‐ligand systems it appears to be ideally suited as a general tool to anchor ligands to AFM tips for recognition force microscopy/spectroscopy experiments.
Cilia are ubiquitous cell surface projections that mediate various sensory- and motility-based processes and are implicated in a growing number of multi-organ genetic disorders termed ciliopathies. To identify new components required for cilium biogenesis and function, we sought to further define and validate the transcriptional targets of DAF-19, the ciliogenic C. elegans RFX transcription factor. Transcriptional profiling of daf-19 mutants (which do not form cilia) and wild-type animals was performed using embryos staged to when the cell types developing cilia in the worm, the ciliated sensory neurons (CSNs), still differentiate. Comparisons between the two populations revealed 881 differentially regulated genes with greater than a 1.5-fold increase or decrease in expression. A subset of these was confirmed by quantitative RT-PCR. Transgenic worms expressing transcriptional GFP fusions revealed CSN-specific expression patterns for 11 of 14 candidate genes. We show that two uncharacterized candidate genes, termed dyf-17 and dyf-18 because their corresponding mutants display dye-filling (Dyf) defects, are important for ciliogenesis. DYF-17 localizes at the base of cilia and is specifically required for building the distal segment of sensory cilia. DYF-18 is an evolutionarily conserved CDK7/CCRK/LF2p-related serine/threonine kinase that is necessary for the proper function of intraflagellar transport, a process critical for cilium biogenesis. Together, our microarray study identifies targets of the evolutionarily conserved RFX transcription factor, DAF-19, providing a rich dataset from which to uncover—in addition to DYF-17 and DYF-18—cellular components important for cilium formation and function.
In rat brain, high-conductance Ca2+-activated K+ (BK) channels are targeted to axons and nerve terminals [Knaus, H. G., et al. (1996) J. Neurosci. 16, 955-963], but absolute levels of their regional expression and subunit composition have not yet been fully established. To investigate these issues, an IbTX analogue ([125I]IbTX-D19Y/Y36F) was employed that selectively binds to neuronal BK channels with high affinity (Kd = 21 pM). Cross-linking experiments with [125I]IbTX-D19Y/Y36F in the presence of a bifunctional reagent led to covalent incorporation of radioactivity into a protein with an apparent molecular mass of 25 kDa. Deglycosylation and immunoprecipitation studies with antibodies raised against alpha- and smooth muscle beta-subunits of the BK channel suggest that the beta-subunit that is associated with the neuronal BK channel is a novel protein. Quantitative receptor autoradiography reveals the highest levels of BK channel expression in the outer layers of the neocortex, hippocampal perforant path projections, and the interpeduncular nucleus. This distribution pattern has also been confirmed in immunocytochemical experiments with a BK channel-selective antibody. Taken together, these findings imply that neuronal BK channels exhibit a restricted distribution in brain and have a subunit composition different from those of their smooth muscle congeners.
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