BK Channels Control Cerebellar Purkinje and Golgi Cell Rhythmicity In Vivo

Calcium signaling plays a central role in normal CNS functioning and dysfunction. As cerebellar Purkinje cells express the major regulatory elements of calcium control and represent the sole integrative output of the cerebellar cortex, changes in neural activity- and calcium-mediated membrane properties of these cells are expected to provide important insights into both intrinsic and network physiology of the cerebellum. We studied the electrophysiological behavior of Purkinje cells in genetically engineered alert mice that do not express BK calcium-activated potassium channels and in wild-type mice with pharmacological BK inactivation. We confirmed BK expression in Purkinje cells and also demonstrated it in Golgi cells. We demonstrated that either genetic or pharmacological BK inactivation leads to ataxia and to the emergence of a beta oscillatory field potential in the cerebellar cortex. This oscillation is correlated with enhanced rhythmicity and synchronicity of both Purkinje and Golgi cells. We hypothesize that the temporal coding modification of the spike firing of both Purkinje and Golgi cells leads to the pharmacologically or genetically induced ataxia

Deletion of the Ca2+-activated potassium (BK) alpha-subunit but not the BK-beta-1-subunit leads to progressive hearing loss

The large conductance voltage- and Ca2+-activated potassium (BK) channel has been suggested to play an important role in the signal transduction process of cochlear inner hair cells. BK channels have been shown to be composed of the pore-forming alpha-subunit coexpressed with the auxiliary beta-1-subunit. Analyzing the hearing function and cochlear phenotype of BK channel alpha-(BKalpha–/–) and beta-1-subunit (BKbeta-1–/–) knockout mice, we demonstrate normal hearing function and cochlear structure of BKbeta-1–/– mice. During the first 4 postnatal weeks also, BKalpha–/– mice most surprisingly did not show any obvious hearing deficits. High-frequency hearing loss developed in BKalpha–/– mice only from ca. 8 weeks postnatally onward and was accompanied by a lack of distortion product otoacoustic emissions, suggesting outer hair cell (OHC) dysfunction. Hearing loss was linked to a loss of the KCNQ4 potassium channel in membranes of OHCs in the basal and midbasal cochlear turn, preceding hair cell degeneration and leading to a similar phenotype as elicited by pharmacologic blockade of KCNQ4 channels. Although the actual link between BK gene deletion, loss of KCNQ4 in OHCs, and OHC degeneration requires further investigation, data already suggest human BK-coding slo1 gene mutation as a susceptibility factor for progressive deafness, similar to KCNQ4 potassium channel mutations. © 2004, The National Academy of Sciences. Freely available online through the PNAS open access option

Osteopenia Due to Enhanced Cathepsin K Release by BK Channel Ablation in Osteoclasts

BACKGROUND: The process of bone resorption by osteoclasts is regulated by Cathepsin K, the lysosomal collagenase responsible for the degradation of the organic bone matrix during bone remodeling. Recently, Cathepsin K was regarded as a potential target for therapeutic intervention of osteoporosis. However, mechanisms leading to osteopenia, which is much more common in young female population and often appears to be the clinical pre-stage of idiopathic osteoporosis, still remain to be elucidated, and molecular targets need to be identified. METHODOLOGY/PRINCIPAL FINDINGS: We found, that in juvenile bone the large conductance, voltage and Ca(2+)-activated (BK) K(+) channel, which links membrane depolarization and local increases in cytosolic calcium to hyperpolarizing K(+) outward currents, is exclusively expressed in osteoclasts. In juvenile BK-deficient (BK(-/-)) female mice, plasma Cathepsin K levels were elevated two-fold when compared to wild-type littermates. This increase was linked to an osteopenic phenotype with reduced bone mineral density in long bones and enhanced porosity of trabecular meshwork in BK(-/-) vertebrae as demonstrated by high-resolution flat-panel volume computed tomography and micro-CT. However, plasma levels of sRANKL, osteoprotegerin, estrogene, Ca(2+) and triiodthyronine as well as osteoclastogenesis were not altered in BK(-/-) females. CONCLUSION/SIGNIFICANCE: Our findings suggest that the BK channel controls resorptive osteoclast activity by regulating Cathepsin K release. Targeted deletion of BK channel in mice resulted in an osteoclast-autonomous osteopenia, becoming apparent in juvenile females. Thus, the BK(-/-) mouse-line represents a new model for juvenile osteopenia, and revealed the BK channel as putative new target for therapeutic controlling of osteoclast activity

Dual role of protein kinase C on BK channel regulation

Large conductance voltage- and Ca2+-activated potassium channels (BK channels) are important feedback regulators in excitable cells and are potently regulated by protein kinases. The present study reveals a dual role of protein kinase C (PKC) on BK channel regulation. Phosphorylation of S695 by PKC, located between the two regulators of K+ conductance (RCK1/2) domains, inhibits BK channel open-state probability. This PKC-dependent inhibition depends on a preceding phosphorylation of S1151 in the C terminus of the channel α-subunit. Phosphorylation of only one α-subunit at S1151 and S695 within the tetrameric pore is sufficient to inhibit BK channel activity. We further detected that protein phosphatase 1 is associated with the channel, constantly counteracting phosphorylation of S695. PKC phosphorylation at S1151 also influences stimulation of BK channel activity by protein kinase G (PKG) and protein kinase A (PKA). Though the S1151A mutant channel is activated by PKA only, the phosphorylation of S1151 by PKC renders the channel responsive to activation by PKG but prevents activation by PKA. Phosphorylation of S695 by PKC or introducing a phosphomimetic aspartate at this position (S695D) renders BK channels insensitive to the stimulatory effect of PKG or PKA. Therefore, our findings suggest a very dynamic regulation of the channel by the local PKC activity. It is shown that this complex regulation is not only effective in recombinant channels but also in native BK channels from tracheal smooth muscle

LFPO in BK^−/− mice is highly synchronized along the frontal and sagittal plane.

<p>(<i>A–B</i>) Simultaneous recordings of two LFPO with electrodes at a distance of 400 µm apart along the frontal (A) and sagittal (B) planes. (<i>C–D</i>) Cross-correlation function (CCF) of the recorded signals illustrated in A and B. (<i>E–F</i>) Plotted values of the maximal CCF coefficient and the corresponding distance between recording electrodes in a same BK^−/− mouse in the frontal (E) and in the sagittal (F) plane. Note the absence of significant variation.</p

Cerebellar cortices of BK^−/− mice present a LFPO in the beta-range phase-locked with both the simple and complex spikes.

<p>(<i>A–B</i>) Simultaneous recording of a LFPO and a Purkinje cell (250 µm-apart along the parallel fiber axis) and Fast-Fourier-Transform of the LFPO. (<i>C–F</i>). Spike-trigger averaging of the LFPO using the complex (C–D) and the simple (E–F) spike. Note the phase-difference in the phase-locking of complex and simple spikes. The smoother aspect of the simple spike triggered wave is due to the much greater number of triggering spikes. Traces D and F are low-pass filtered (<500 Hz); note the difference in time scale. Arrows indicate the time lag. (<i>G</i>) Simple spike autocorrelogram of the Purkinje cell illustrated in A. Arrow indicates the correspondence between low frequency rhythmicity and LFPO wave. (<i>H</i>) Cross-correlation function between the non-filtered simple and complex spike triggered averaging, confirming the time lag around 7 ms.</p

Intracerebellar microinjection of paxilline in WT mice reproduces the rhythmic firing of Purkinje cells and the ataxic behavior of BK^−/− mice.

<p>(<i>A</i>,<i>B</i>) Spontaneous firing of a Purkinje cell recorded in a WT mouse (<i>A</i>) and corresponding autocorrelogram (<i>B</i>). Note the absence of rhythmicity. (<i>C</i>,<i>D</i>) The same, following microinjection of paxilline. (<i>E–H</i>) Bar graphs of Purkinje cells simple spike rhythmicity (n = 13)(<i>E</i>) and frequency (n = 13)(<i>F</i>), Purkinje cells complex spike frequency (n = 8)(<i>G</i>) and subsequent pause duration (n = 8) (<i>H</i>) before and after paxilline injection and in BK^−/− (n = 48, value illustrated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0007991#pone-0007991-g002" target="_blank">fig 2</a> and reproduced here for comparison purpose). Stars indicate significance as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0007991#pone-0007991-g002" target="_blank">fig 2</a>, for student t test for paired values (comparison between before and after injection) and unpaired values (comparison between WT PC after injection and PC in BK^−/−) (<i>I</i>,<i>J</i>) Runway test, bar graph of mean number of slips (<i>I</i>) and time to reach the end of the bar (<i>J</i>) before and after paxilline injection (n = 9).</p