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KCNMA1 is a gene located at 10q22 that encodes the pore-forming &alpha, subunit of the large-conductance Ca2+-activated K+ channel. KCNMA1 is down-regulated in gastric carcinoma tumors, through hypermethylation of its promoter. In the present study, we have evaluated the expression levels of KCNMA1 both in a mouse model of Colorectal Cancer (CRC) and in human CRC samples. Additionally, epigenetic mechanisms of KCNMA1 gene regulation were investigated. We observed a significant down-regulation of KCNMA1 both in a human and mouse model of CRC. No differences in KCNMA1 levels were, however, observed at different TNM stages. We also wanted to determine whether the modulation in KCNMA1 was dependent on epigenetic mechanisms. A statistically significant inverse correlation between KCNMA1 expression and mir-17-5p levels was observed in patients with CRC. Furthermore, in the tumor samples, we found a significant hypermethylation of the promoter, in the loci cg24113782 and cg25655799, compared to healthy tissue. Overall, our data suggest the possible use of KCNMA1 as a therapeutic target in the early stages of CRC.

In the last 15 years, remarkable progress has been realized in identifying the genes that encode the ion-transporting proteins involved in exocrine gland function, including salivary glands. Among these proteins, Ca(2+)-dependent K(+) channels take part in key functions including membrane potential regulation, fluid movement and K(+) secretion in exocrine glands. Two K(+) channels have been identified in exocrine salivary glands: (1) a Ca(2+)-activated K(+) channel of intermediate single channel conductance encoded by the KCNN4 gene, and (2) a voltage- and Ca(2+)-dependent K(+) channel of large single channel conductance encoded by the KCNMA1 gene. This review focuses on the physiological roles of Ca(2+)-dependent K(+) channels in exocrine salivary glands. We also discuss interesting recent findings on the regulation of Ca(2+)-dependent K(+) channels by protein-protein interactions that may significantly impact exocrine gland physiology.

Expression and activation of the large conductance calcium and voltage-gated potassium (BK Ca ) channels encoded by Kcnma1 gene is shown to be vital in cardioprotection from ischemia-reperfusion injury. BK Ca channels present in SA node cells regulate the heart rate, and in blood vessels play an active role in vascular relaxation. However, the role of BK Ca in regulation of structure and function of the heart is not fully-established. Using Kcnma1 -/- mice, we have observed structural changes in cardiomyocytes and compromised cardiac function as compared to wild type mice. Absence of BK Ca resulted in significant increase in size of adult cardiomyocytes (from 7.95 + 0.1 um 2 to 9.68 + 0.1 um 2 , p < 0.01, n=480 cells each) and also increased cardiac fibrosis. Further to determine underlying signaling mechanisms in cardiac hypertrophy, we performed microarray analysis of RNAs isolated from wild type and Kcnma1 -/- mice (n=3) hearts. We found up regulation of a class of cardiac hypertrophy markers (myosin variants) and changes in the expression of several mitochondrial genes (such as ND4) directly associated with heart diseases in Kcnma1 -/- mice. To evaluate the functional consequence of absence of BK Ca , we performed high-resolution echocardiography on wild type and Kcnma1 -/- mice. Under anesthesia (1.5% isoflurane), left ventricle of Kcnma1 -/- mice showed significant reduction (p < 0.05) in ejection fraction (56 + 2 %, n=7) as compared to wild type (74 + 3 %, n=6) as well as fractional shortening (23 + 3 %, n=7, and 39 + 3 %, n=6, respectively). Similarly, right ventricle had a lower ejection fraction (35.7 + 4% vs 56.9 + 5 %, n > 5) in Kcnma1 -/- as compared to wild type mice. In agreement with our histopathology and microarray data, Kcnma1 -/- mice showed increased posterior wall thickness (0.75 + 0.3 mm vs 0.62 + 0.1 mm) and interventricular septum thickness (0.83 + 0.4 mm, n=7 vs 0.68 + 0.3 mm, n=6) . Together, these data imply that BK Ca plays a direct role in cardiac hypertrophy and cardiac function.

Functional properties of large conductance Ca(2+) activated potassium (BK) channels are determined by complex alternative splicing of the Kcnma1 gene encoding the alpha pore-forming subunit. Inclusion of the STREX exon in a C-terminal splice site is dynamically regulated and confers enhanced Ca(2+) sensitivity and channel inhibition via cAMP-dependent phosphorylation. Here, we describe a real time quantitative PCR (qPCR) approach to investigate relative changes in the expression of STREX and ZERO splice variants using a newly designed set of probes and primers for TaqMan-based qPCR analysis of cDNA from the rat dentate gyrus at different time points following pilocarpine-induced status epilepticus. Reduction in Kcnma1 gene expression is associated with a relative increase of STREX splice variant. Relative expression of STREX variant mRNA was increased at 10 days and at more than 1 month following status epilepticus. The biological consequences of seizure-related changes in alternative splicing of Kcnma1 deserve additional investigation.

Expression and activation of BKCa encoded by a Kcnma1 gene, is known to play a direct role in cardioprotection against ischemia-reperfusion injury by modulating mitochondrial function, and in regulating the heart rate. However, the role of BKCa in cardiac function is not yet deciphered Cardiac

Mitochondria are known for their unique membrane architecture with the inner membrane folded into cristae. It is also known for long that mitochondria undergo drastic changes in volume. These volume changes depend on potassium fluxes. Influx via mitochondrial potassium channels (e.g. mitoBKCa or mitoKATP) results in swelling of mitochondria and unfolding of the cristae, which could be potentially translated into changes in the inner membrane tension. However, little is known about mechanobiology of mitochondria including existence of mechanosensitive channels.Mitochondrial large conductance calcium activated potassium channel BKCa (mitoBKCa) resides in the inner mitochondrial membrane (IMM) and plays an important role in cytoprotection. KCNMA1 gene encodes for both mitochondrial and plasmalemmal versions of BKCa channels, which share common features including large conductance, synergistic gating by voltage and Ca2+ ions. However, it was suggested that one of plasmalemmal splice variants of BKCa containing STREX exon was uniquely mechanosensitive. Here we show that mitoBKCa formed by another splice variant BKCa-DEC is also sensitive to stretch. We discuss potential physiological role of mechanosensitivity of mitoBKCa during mitochondria swelling.

Alcohol abuse is a significant public health problem. Understanding the molecular effects of ethanol is important for the identification of at risk individuals, as well as the development of novel pharmacotherapies. The large conductance calcium sensitive potassium (BK) channel has emerged as an important player in the behavioral response to ethanol in genetic studies in several model organisms and in humans. The BK channel, slo-1, was identified in a forward genetics screen as a major ethanol target in C. elegans for the effects of ethanol on locomotion and egg-laying behaviors. Regulation of the expression of the BK channel, slo, in Drosophila underlies the development of rapid tolerance to ethanol and benzyl alcohol sedation. Rodent expression studies of the BK-encoding KCNMA1 gene have identified regulation of mRNA levels in response to ethanol exposure, and knock out studies in mice have demonstrated that the beta subunits of the BK channel, KCNMB1 and KCNMB4, can modulate ethanol sensitivity of the channel in electrophysiological preparations, and can influence drinking behavior. In human genetics studies, both KCNMA1 and the genes encoding beta subunits of the BK channel have been associated with alcohol dependence. This review describes the genetic data for a role for BK channels in mediating behavioral responses to ethanol across these species.

The large-conductance Ca 2+ - and voltage-activated K + channel (BK Ca, MaxiK), which is encoded by the Kcnma1 gene, is generally expressed at the plasma membrane of excitable and nonexcitable cells. However, in adult cardiomyocytes, a BK Ca -like channel activity has been reported in the mitochondria but not at the plasma membrane. The putative opening of this channel with the BK Ca agonist, NS1619, protects the heart from ischemic insult. However, the molecular origin of mitochondrial BK Ca (mitoBK Ca ) is unknown because its linkage to Kcnma1 has been questioned on biochemical and molecular grounds. Here, we unequivocally demonstrate that the molecular correlate of mitoBK Ca is the Kcnma1 gene, which produces a protein that migrates at ∼140 kDa and arranges in clusters of ∼50 nm in purified mitochondria. Physiological experiments further support the origin of mitoBK Ca as a Kcnma1 product because NS1619-mediated cardioprotection was absent in Kcnma1 knockout mice. Finally, BK Ca transcript analysis and expression in adult cardiomyocytes led to the discovery of a 50-aa C-terminal splice insert as essential for the mitochondrial targeting of mitoBK Ca .

Background The prognosis for patients with breast tumor metastases to brain is extremely poor. Identification of prognostic molecular markers of the metastatic process is critical for designing therapeutic modalities for reducing the occurrence of metastasis. Although ubiquitously present in most human organs, large-conductance calcium- and voltage-activated potassium channel (BKCa) channels are significantly upregulated in breast cancer cells. In this study we investigated the role of KCNMA1 gene that encodes for the pore-forming α-subunit of BKCa channels in breast cancer metastasis and invasion. Methods We performed Global exon array to study the expression of KCNMA1 in metastatic breast cancer to brain, compared its expression in primary breast cancer and breast cancers metastatic to other organs, and validated the findings by RT-PCR. Immunohistochemistry was performed to study the expression and localization of BKCa channel protein in primary and metastatic breast cancer tissues and breast cancer cell lines. We performed matrigel invasion, transendothelial migration and membrane potential assays in established lines of normal breast cells (MCF-10A), non-metastatic breast cancer (MCF-7), non-brain metastatic breast cancer cells (MDA-MB-231), and brain-specific metastatic breast cancer cells (MDA-MB-361) to study whether BKCa channel inhibition attenuates breast tumor invasion and metastasis using KCNMA1 knockdown with siRNA and biochemical inhibition with Iberiotoxin (IBTX). Results The Global exon array and RT-PCR showed higher KCNMA1 expression in metastatic breast cancer in brain compared to metastatic breast cancers in other organs. Our results clearly show that metastatic breast cancer cells exhibit increased BKCa channel activity, leading to greater invasiveness and transendothelial migration, both of which could be attenuated by blocking KCNMA1. Conclusion Determining the relative abundance of BKCa channel expression in breast cancer metastatic to brain and the mechanism of its action in brain metastasis will provide a unique opportunity to identify and differentiate between low grade breast tumors that are at high risk for metastasis from those at low risk for metastasis. This distinction would in turn allow for the appropriate and efficient application of effective treatments while sparing patients with low risk for metastasis from the toxic side effects of chemotherapy.

International audience; Objective: Autism is a complex, largely genetic psychiatric disorder. In the majority of cases, the cause of autism is not known, but there is strong evidence for a genetic etiology. To identify candidate genes, the physical mapping of balanced chromosomal aberrations is a powerful strategy, since several genes have been characterized in numerous disorders. In this study, the authors analyzed a balanced reciprocal translocation arising de novo in a subject with autism and mental retardation. Method: The authors performed the physical mapping of the balanced 9q23/ 10q22 translocation by fluorescent in situ hybridization experiments using bacterial artificial chromosome clones covering the areas of interest. Results: Findings revealed that the KCNMA1 gene, which encodes the alpha- subunit of the large conductance Ca2+-activated K+ ( BKCa) channel, a synaptic regulator of neuronal excitability, is physically disrupted. Further molecular and functional analyses showed the haploinsufficiency of this gene as well as decreased activity of the coded BKCa channel. This activity can be enhanced in vitro by addition of a BKCa channel opener (BMS-204352). Further mutational analyses on 116 autistic subjects led to the identification of an amino acid substitution located in a highly conserved domain of the protein not found in comparison subjects. Conclusions: These results suggest a possible association between a functional defect of the BKCa channel and autistic disorder and raise the hypothesis that deficits in synaptic transmission may contribute to the physiopathology of autism and mental deficiency.

Abstract #2043 Background: The prognosis for patients with brain metastasis from breast cancer is extremely poor. A significant number of breast tumor patients despite aggressive therapy develop metastatic disease in brain. The mechanism or the cause of breast cancer metastasis to brain is unknown. Therefore, it is critical to identify prognostic molecular markers of the metastatic process in order to design therapeutic modalities for reducing the occurrence of metastasis. In this study we investigated KCNMA1 gene, which encodes alpha subunit of calcium activated potassium (KCa) channels. Altered KCa channels, are described in a wide variety of tumor cell types and up-regulation of KCa channels was shown to be a novel mechanism for the malignant phenotype of human tumor cells. Material and Methods: We performed Global GeneChip exon arrays to study the expression of KCNMA1 gene in metastatic breast cancer in brain and compared it with primary breast cancer and breast cancers metastatic to other organs, and validated the findings by RT-PCR. Immuno-histochemistry was performed to study the expression and localization of alpha subunit of KCa channel protein in primary and metastatic breast tumor tissues and breast cancer cell lines. We performed Matrigel invasion, transendothelial migration and membrane potential assays in normal breast cells (MCF-10A), non-metastatic breast cancer (MCF-7), non-brain metastatic breast cancer cells (MDA-MB-231), and brain-specific metastatic breast cancer cells (MDA-MB-361) to study whether KCa channel inhibition attenuates breast tumor invasion and metastasis to brain. Results: The Global GeneChip exon array and RT-PCR showed higher KCNMA1 gene expression in metastatic breast cancer in brain compared to metastatic breast cancers in other organs. Our results clearly show that metastatic breast cancer cells exhibit increased KCa channel activity, leading to greater invasion and trans-endothelial migration, which could be attenuated by blocking the KCNMA1 gene with siRNA and biochemical inhibition with IBTX. Discussion: Recent investigations in patients with metastatic lung and breast cancers reported alarming rates of brain metastases. Unraveling the genetic aberrations that drive the metastatic events in these cancer cells offer insights into how to limit or prevent this lethal process. For instance, determining the relative abundance of KCNMA1 gene in breast cancer metastasis to brain and understanding the mechanism of brain metastasis provides a unique opportunity to identify specific genetic markers that could differentiate between low grade breast tumors that are at high risk for metastasis from those at low risk for metastasis. This would in turn allow for the appropriate and efficient application of treatments to obtain the greatest benefit while sparing low risk patients for metastasis from the toxic side effects of chemotherapy. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 2043.