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 crossactivation 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.

Malfunctions of potassium channels are increasingly implicated as causes of neurological disorders. However, the functional roles of the large-conductance voltage-and Ca 2؉ -activated K ؉ channel (BK channel), a unique calcium, and voltage-activated potassium channel type have remained elusive. Here we report that mice lacking BK channels (BK ؊/؊ ) show cerebellar dysfunction in the form of abnormal conditioned eye-blink reflex, abnormal locomotion and pronounced deficiency in motor coordination, which are likely consequences of cerebellar learning deficiency. At the cellular level, the BK ؊/؊ mice showed a dramatic reduction in spontaneous activity of the BK ؊/؊ cerebellar Purkinje neurons, which generate the sole output of the cerebellar cortex and, in addition, enhanced short-term depression at the only output synapses of the cerebellar cortex, in the deep cerebellar nuclei. The impairing cellular effects caused by the lack of postsynaptic BK channels were found to be due to depolarization-induced inactivation of the action potential mechanism. These results identify previously unknown roles of potassium channels in mammalian cerebellar function and motor control. In addition, they provide a previously undescribed animal model of cerebellar ataxia. P otassium channels are the largest and most diverse class of ion channels underlying electrical signaling in the brain (1). By causing highly regulated, time-dependent, and localized polarization of the cell membrane, the opening of K ϩ channels mediates feedback control of excitability in a variety of cell types and conditions (1). Consequently, K ϩ channel dysfunctions can cause a range of neurological disorders (2-6), and drugs that target K ϩ channels hold promise for a variety of clinical applications (7).Among the wide range of voltage-and calcium-gated K ϩ channel types, one stands out as unique: the large-conductance voltage-and Ca 2ϩ -activated K ϩ channel (BK channel, also termed Slo or Maxi-K) differs from all other K ϩ channels in that it can be activated by both intracellular Ca 2ϩ ions and membrane depolarization (8). These channels are widely expressed in central and peripheral neurons, as well as in other tissues (9), and are regarded as a promising drug target (10). However, the functions of the BK channels in vivo have not previously been directly tested in any vertebrate species. We therefore decided to examine the functions of these channels by inactivating the gene encoding the pore-forming channel protein. MethodsA complete description of the methods is given in Supporting Methods, which is published as supporting information on the PNAS web site.Generation of BK Channel ␣ Subunit-Deficient Mice. In the targeting vector (Fig. 5, which is published as supporting information on the PNAS web site), the pore exon was flanked by a single loxP site and a floxed neo͞tk cassette. Correctly targeted embryonic stem cells were injected into C57BL͞6 blastocysts and resulting chimeric mice mated with C57BL͞6. Homozygous BK-deficient mice (F 2 generation) ...

Calcium release from the endoplasmic reticulum controls a number of cellular processes, including proliferation and contraction of smooth muscle and other cells. Calcium release from inositol 1,4,5-trisphosphate (IP3)-sensitive stores is negatively regulated by binding of calmodulin to the IP3 receptor (IP3R) and the NO/cGMP/cGMP kinase I (cGKI) signalling pathway. Activation of cGKI decreases IP3-stimulated elevations in intracellular calcium, induces smooth muscle relaxation and contributes to the antiproliferative and pro-apoptotic effects of NO/cGMP. Here we show that, in microsomal smooth muscle membranes, cGKIbeta phosphorylated the IP3R and cGKIbeta, and a protein of relative molecular mass 125,000 which we now identify as the IP3R-associated cGMP kinase substrate (IRAG). These proteins were co-immunoprecipitated by antibodies directed against cGKI, IP3R or IRAG. IRAG was found in many tissues including aorta, trachea and uterus, and was localized perinuclearly after heterologous expression in COS-7 cells. Bradykinin-stimulated calcium release was not affected by the expression of either IRAG or cGKIbeta, which we tested in the absence and presence of cGMP. However, calcium release was inhibited after co-expression of IRAG and cGKIbeta in the presence of cGMP. These results identify IRAG as an essential NO/cGKI-dependent regulator of IP3-induced calcium release.

Abstract-Both cGMP-dependent and -independent mechanisms have been implicated in the regulation of vascular tone by NO. We analyzed acetylcholine (ACh)-and NO-induced relaxation in pressurized small arteries and aortic rings from wild-type (wt) and cGMP kinase I-deficient (cGKI -/-) mice. Low concentrations of NO and ACh decreased the spontaneous myogenic tone in wt but not in cGKI -/-arteries. However, contractions of cGKI -/-arteries and aortic rings were reduced by high concentrations (10 mol/L) of 2-(N,N-diethylamino)-diazenolate-2-oxide (DEA-NO). Iberiotoxin, a specific blocker of Ca 2ϩ -activated K ϩ (BK Ca ) channels, only partially prevented the relaxation induced by DEA-NO or ACh in pressurized vessels and aortic rings. DEA-NO increased the activity of BK Ca channels only in vascular smooth muscle cells isolated from wt cGKI ϩ/ϩ mice. These results suggest that low physiological concentrations of NO decrease vascular tone through activation of cGKI, whereas high concentrations of DEA-NO relax vascular smooth muscle independent of cGKI and BK Ca . NO-stimulated, cGKI-independent relaxation was antagonized by the inhibition of soluble guanylyl cyclase or cAMP kinase (cAK). DEA-NO increased cGMP to levels that are sufficient to activate cAK. cAMP-dependent relaxation was unperturbed in cGKI -/-vessels. In conclusion, low concentrations of NO relax vessels by activation of cGKI, whereas in the absence of cGKI, NO can relax small and large vessels by cGMP-dependent activation of cAK. T he NO/cGMP signaling cascade plays an essential role in vascular smooth muscle relaxation, and clinical studies indicate that endothelium-derived NO is involved in normal and pathological blood pressure regulation in humans. 1 Targeted inactivation of endothelial NO synthase, 2,3 atrial natriuretic peptide (ANP), 4 and the ANP receptor guanylyl cyclase-A 5,6 causes hypertension. Animal and in vitro studies also show that NO and ANP decreases blood pressure by relaxation of small arteries and arterioles. 7 In most studies, this decrease in vascular tone was associated with increases in cGMP. Some studies suggested that NO reduces vascular tone independent of cGMP and cGMP kinase I (cGKI). 8 -11 The mechanisms that are activated by NO or cGMP in smooth muscle are controversial. [12][13][14][15][16] The important effector of cGMP, cGKI, is highly expressed in smooth muscle. Deletion of the gene for cGKI can led to multiple phenotypes, including high blood pressure in young mice. 17 Interestingly, and in contrast to previous suggestions, 13 adenosine A 2 receptor-and cAMP-dependent relaxation of aortic rings was not affected by the lack of cGKI. 17 Older cGKI-deficient (cGKI -/-) mice, many of which have multiple infections, have normal or only slightly elevated blood pressure, 17 suggesting that cGKI is not absolutely required to lower vascular tone and can be bypassed. A potential alternative pathway could be an increased production of endothelium-derived hyperpolarizing factors (EDHF). 18 Activation of large and small K Ca ch...