Matthias Sausbier

Defective smooth muscle regulation in cGMP kinase I‐deficient mice

Regulation of smooth muscle contractility is essential for many important biological processes such as tissue perfusion, cardiovascular haemostasis and gastrointestinal motility. While an increase in calcium initiates smooth muscle contraction, relaxation can be induced by cGMP or cAMP. cGMP‐dependent protein kinase I (cGKI) has been suggested as a major mediator of the relaxant effects of both nucleotides. To study the biological role of cGKI and its postulated cross‐activation by cAMP, we inactivated the gene coding for cGKI in mice. Loss of cGKI abolishes nitric oxide (NO)/cGMP‐dependent relaxation of smooth muscle, resulting in severe vascular and intestinal dysfunctions. However, cGKI‐deficient smooth muscle responded normally to cAMP, indicating that cAMP and cGMP signal via independent pathways, with cGKI being the specific mediator of the NO/cGMP effects in murine smooth muscle.

Elevated Blood Pressure Linked to Primary Hyperaldosteronism and Impaired Vasodilation in BK Channel–Deficient Mice

Background—Abnormally elevated blood pressure is the most prevalent risk factor for cardiovascular disease. The large-conductance, voltage- and Ca2+-dependent K+ (BK) channel has been proposed as an important effector in the control of vascular tone by linking membrane depolarization and local increases in cytosolic Ca2+ to hyperpolarizing K+ outward currents. However, the BK channel may also affect blood pressure by regulating salt and fluid homeostasis, particularly by adjusting the renin-angiotensin-aldosterone system. Methods and Results—Here we report that deletion of the pore-forming BK channel &agr; subunit leads to a significant blood pressure elevation resulting from hyperaldosteronism accompanied by decreased serum K+ levels as well as increased vascular tone in small arteries. In smooth muscle from small arteries, deletion of the BK channel leads to a depolarized membrane potential, a complete lack of membrane hyperpolarizing spontaneous K+ outward currents, and an attenuated cGMP vasorelaxation associated with a reduced suppression of Ca2+ transients by cGMP. The high level of BK channel expression observed in wild-type adrenal glomerulosa cells, together with unaltered serum renin activities and corticotropin levels in mutant mice, suggests that the hyperaldosteronism results from abnormal adrenal cortical function in BK−/− mice. Conclusions—These results identify previously unknown roles of BK channels in blood pressure regulation and raise the possibility that BK channel dysfunction may underlie specific forms of hyperaldosteronism.

Ca2+ -activated K+ channels of the BK-type in the mouse brain.

An antibody against the 442 carboxy-terminal amino acids of the BK channel α-subunit detects high immunoreactivity within the telencephalon in cerebral cortices, olfactory bulb, basal ganglia and hippocampus, while lower levels are found in basal forebrain regions and amygdala. Within the diencephalon, high density was found in nuclei of the ventral and dorsal thalamus and the medial habenular nucleus, and low density in the hypothalamus. The fasciculus retroflexus and its termination in the mesencephalic interpeduncular nucleus are prominently stained. Other mesencephalic expression sites are periaquaeductal gray and raphe nuclei. In the rhombencephalon, BK channels are enriched in the cerebellar cortex and in the locus coeruleus. Strong immunoreactivity is also contained in the vestibular nuclei, but not in cranial nerves and their intramedullary course of their roots. On the cellular level, BK channels show pre- and postsynaptic localizations, i.e., in somata, dendrites, axons and synaptic terminals.

Distal Colonic K+ Secretion Occurs via BK Channels

K(+) secretion in the kidney and distal colon is a main determinant of K(+) homeostasis. This study investigated the identity of the relevant luminal secretory K(+) ion channel in distal colon. An Ussing chamber was used to measure ion transport in the recently generated BK channel-deficient (BK(-/-)) mice. BK(-/-) mice display a significant colonic epithelial phenotype with (1) lack of Ba(2+)-sensitive resting K(+) secretion, (2) absence of K(+) secretion stimulated by luminal P2Y(2) and P2Y(4) receptors, (3) absence of luminal Ca(2+) ionophore (A23187)-stimulated K(+) secretion, (4) reduced K(+) and increased Na(+) contents in feces, and (5) an increased colonic Na(+) absorption. In contrast, resting and uridine triphosphate (UTP)-stimulated K(+) secretion was not altered in mice that were deficient for the intermediate conductance Ca(2+)-activated K(+) channel SK4. BK channels localize to the luminal membrane of crypt, and reverse transcription-PCR results confirm the expression of the BK channel alpha-subunit in isolated distal colonic crypts. It is concluded that BK channels are the responsible K(+) channels for resting and stimulated Ca(2+)-activated K(+) secretion in mouse distal colon.

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.

Two classes of outer hair cells along the tonotopic axis of the cochlea

The molecular basis of high versus low frequency hearing loss and the differences in the sensitivity of outer hair cells depending on their cochlear localization are currently not understood. Here we demonstrate the existence of two different outer hair cell phenotypes along the cochlear axis. Outer hair cells in low frequency regions exhibit early sensitivity for loss of Ca(v)1.3 (alpha1 subunit 1.3 forming the class D L-type voltage-gated Ca(2+) channel), while high frequency regions display a progressive susceptibility for loss of the Ca(2+)-activated large conductance K(+) (BK) channel. Despite deafness, young Ca(v)1.3-deficient mice displayed distortion-product otoacoustic emissions (DPOAEs), indicating functional outer hair cells in the higher frequency range of the cochlea. Considering that DPOAEs are also found in the human deafness syndrome DFNB9 caused by mutations in the synaptic vesicle protein otoferlin, we tested the expression of otoferlin in outer hair cells. Surprisingly, otoferlin showed a distinct tonotopic expression pattern at both the mRNA and protein level. Otoferlin-expressing, Ca(v)1.3 deletion-sensitive outer hair cells in the low frequency range could be clearly separated from otoferlin-negative, BK deletion-sensitive outer hair cells in the high frequency range. In addition, BK deletion led to a higher noise vulnerability in low frequency regions, which are normally unaffected by the BK deletion alone, suggesting that BK currents are involved in survival mechanisms of outer hair cells under noise conditions. Our findings propose new mechanisms and candidate genes for explaining high and low frequency hearing loss.

Blunted IgE-Mediated Activation of Mast Cells in Mice Lacking the Ca2+-Activated K+ Channel KCa3.1

Mast cell stimulation by Ag is followed by the opening of Ca2+-activated K+ channels, which participate in the orchestration of mast cell degranulation. The present study has been performed to explore the involvement of the Ca2+-activated K+ channel KCa3.1 in mast cell function. To this end mast cells have been isolated and cultured from the bone marrow (bone marrow-derived mast cells (BMMCs)) of KCa3.1 knockout mice (KCa3.1−/−) and their wild-type littermates (KCa3.1+/+). Mast cell number as well as in vitro BMMC growth and CD117, CD34, and FcεRI expression were similar in both genotypes, but regulatory cell volume decrease was impaired in KCa3.1−/− BMMCs. Treatment of the cells with Ag, endothelin-1, or the Ca2+ ionophore ionomycin was followed by stimulation of Ca2+-activated K+ channels and cell membrane hyperpolarization in KCa3.1+/+, but not in KCa3.1−/− BMMCs. Upon Ag stimulation, Ca2+ entry but not Ca2+ release from intracellular stores was markedly impaired in KCa3.1−/− BMMCs. Similarly, Ca2+ entry upon endothelin-1 stimulation was significantly reduced in KCa3.1−/− cells. Ag-induced release of β-hexosaminidase, an indicator of mast cell degranulation, was significantly smaller in KCa3.1−/− BMMCs compared with KCa3.1+/+ BMMCs. Moreover, histamine release upon stimulation of BMMCs with endothelin-1 was reduced in KCa3.1−/− cells. The in vivo Ag-induced decline in body temperature revealed that IgE-dependent anaphylaxis was again significantly (by ∼50%) blunted in KCa3.1−/− mice. In conclusion, KCa3.1 is required for Ca2+-activated K+ channel activity and Ca2+-dependent processes such as endothelin-1- or Ag-induced degranulation of mast cells, and may thus play a critical role in anaphylactic reactions.

Disruption of the olivo-cerebellar circuit by Purkinje neuron-specific ablation of BK channels

The large-conductance voltage- and calcium-activated potassium (BK) channels are ubiquitously expressed in the brain and play an important role in the regulation of neuronal excitation. Previous work has shown that the total deletion of these channels causes an impaired motor behavior, consistent with a cerebellar dysfunction. Cellular analyses showed that a decrease in spike firing rate occurred in at least two types of cerebellar neurons, namely in Purkinje neurons (PNs) and in Golgi cells. To determine the relative role of PNs, we developed a cell-selective mouse mutant, which lacked functional BK channels exclusively in PNs. The behavioral analysis of these mice revealed clear symptoms of ataxia, indicating that the BK channels of PNs are of major importance for normal motor coordination. By using combined two-photon imaging and patch-clamp recordings in these mutant mice, we observed a unique type of synaptic dysfunction in vivo, namely a severe silencing of the climbing fiber–evoked complex spike activity. By performing targeted pharmacological manipulations combined with simultaneous patch-clamp recordings in PNs, we obtained direct evidence that this silencing of climbing fiber activity is due to a malfunction of the tripartite olivo-cerebellar feedback loop, consisting of the inhibitory synaptic connection of PNs to the deep cerebellar nuclei (DCN), followed by a projection of inhibitory DCN afferents to the inferior olive, the origin of climbing fibers. Taken together, our results establish an essential role of BK channels of PNs for both cerebellar motor coordination and feedback regulation in the olivo-cerebellar loop.

Aldosterone increases KCa1.1 (BK) channel-mediated colonic K+ secretion

Mammalian K+ homeostasis results from highly regulated renal and intestinal absorption and secretion, which balances the unregulated K+ intake. Aldosterone is known to enhance both renal and colonic K+ secretion. In mouse distal colon K+ secretion occurs exclusively via luminal KCa1.1 (BK) channels. Here we investigate if aldosterone stimulates colonic K+ secretion via BK channels. Luminal Ba2+ and iberiotoxin (IBTX)‐sensitive electrogenic K+ secretion was measured in Ussing chambers. In vivo aldosterone was augmented via a high K+ diet. High K+ diet led to a 2‐fold increase of luminal Ba2+ and IBTX‐sensitive short‐circuit current in distal mouse colonic mucosa. This effect was absent in BK α‐subunit‐deficient (BK−/−) mice. The resting and diet‐induced K+ secretion was stimulated by luminal ionomycin. In BK−/− mice luminal ionomycin did not stimulate K+ secretion. In vitro addition of aldosterone likewise triggered a 2‐fold increase in K+ secretion, which was inhibited by the mineralocorticoid receptor antagonist spironolactone and the BK channel blocker IBTX. Semi‐quantification of mRNA from colonic crypts showed up‐regulation of BK α‐ and β2‐subunits in high K+ diet mice. The BK channel could be detected luminally in colonic crypt cells by immunohistochemistry. The expression level of the channel in the luminal membrane was strongly up‐regulated in K+‐loaded animals. Taken together, these data strongly suggest that aldosterone‐induced K+ secretion occurs via increased expression of luminal BK channels.

The vasodilator 17,18‐epoxyeicosatetraenoic acid targets the pore‐forming BK α channel subunit in rodents

17,18‐Epoxyeicosatetraenoic acid (17,18‐EETeTr) stimulates vascular large‐conductance K+ (BK) channels. BK channels are composed of the pore‐forming BK α and auxiliary BK β1 subunits that confer an increased sensitivity for changes in membrane potential and calcium to BK channels. Ryanodine‐sensitive calcium‐release channels (RyR3) in the sarcoplasmic reticulum (SR) control the process. To elucidate the mechanism of BK channel activation, we performed whole‐cell and perforated‐patch clamp experiments in freshly isolated cerebral and mesenteric artery vascular smooth muscle cells (VSMC) from Sprague–Dawley rats, BK β1 gene‐deficient (−/−), BK α (−/−), RyR3 (−/−) and wild‐type mice. The 17,18‐EETeTr (100 nm) increased tetraethylammonium (1 mm)‐sensitive outward K+ currents in VSMC from wild‐type rats and wild‐type mice. The effects were not inhibited by the epoxyeicosatrienoic acid (EET) antagonist 14,15‐epoxyeicosa‐5(Z)‐enoic acid (10 μm). BK channel currents were increased 3.5‐fold in VSMC from BK β1 (−/−) mice, whereas a 2.9‐fold stimulation was observed in VSMC from RyR3 (−/−) mice (at membrane voltage 60 mV). The effects were similar compared with those observed in cells from wild‐type mice. The BK current increase was neither influenced by strong internal calcium buffering (Ca2+, 100 nm), nor by external calcium influx. The 17,18‐EETeTr did not induce outward currents in VSMC BK α (−/−) cells. We next tested the vasodilator effects of 17,18‐EETeTr on isolated arteries of BK α‐deficient mice. Vasodilatation was largely inhibited in cerebral and mesenteric arteries isolated from BK α (−/−) mice compared with that observed in wild‐type and BK β1 (−/−) arteries. We conclude that 17,18‐EETeTr represents an endogenous BK channel agonist and vasodilator. Since 17,18‐EETeTr is active in small arteries lacking BK β1, the data further suggest that BK α represents the molecular target for the principal action of 17,18‐EETeTr. Finally, the action of 17,18‐EETeTr is not mediated by changes of the internal global calcium concentration or local SR calcium release events.