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Functional coupling of the beta(1) subunit to the large conductance Ca(2+)-activated K(+) channel in the absence of Ca(2+). Increased Ca(2+) sensitivity from a Ca(2+)-independent mechanism.

ABSTRACT: Coexpression of the beta(1) subunit with the alpha subunit (mSlo) of BK channels increases the apparent Ca(2+) sensitivity of the channel. This study investigates whether the mechanism underlying the increased Ca(2+) sensitivity requires Ca(2+), by comparing the gating in 0 Ca(2+)(i) of BK channels composed of alpha subunits to those composed of alpha+beta(1) subunits. The beta(1) subunit increased burst duration approximately 20-fold and the duration of gaps between bursts approximately 3-fold, giving an approximately 10-fold increase in open probability (P(o)) in 0 Ca(2+)(i). The effect of the beta(1) subunit on increasing burst duration was little changed over a wide range of P(o) achieved by varying either Ca(2+)(i) or depolarization. The effect of the beta(1) subunit on increasing the durations of the gaps between bursts in 0 Ca(2+)(i) was preserved over a range of voltage, but was switched off as Ca(2+)(i) was increased into the activation range. The Ca(2+)-independent, beta(1) subunit-induced increase in burst duration accounted for 80% of the leftward shift in the P(o) vs. Ca(2+)(i) curve that reflects the increased Ca(2+) sensitivity induced by the beta(1) subunit. The Ca(2+)-dependent effect of the beta(1) subunit on the gaps between bursts accounted for the remaining 20% of the leftward shift. Our observation that the major effects of the beta(1) subunit are independent of Ca(2+)(i) suggests that the beta(1) subunit mainly alters the energy barriers of Ca(2+)-independent transitions. The changes in gating induced by the beta(1) subunit differ from those induced by depolarization, as increasing P(o) by depolarization or by the beta(1) subunit gave different gating kinetics. The complex gating kinetics for both alpha and alpha+beta(1) channels in 0 Ca(2+)(i) arise from transitions among two to three open and three to five closed states and are inconsistent with Monod-Wyman-Changeux type models, which predict gating among only one open and one closed state in 0 Ca(2+)(i).

SUBMITTER: Nimigean CM

PROVIDER: S-EPMC2232893 | biostudies-literature | 2000 Jun

REPOSITORIES: biostudies-literature

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Functional coupling of the beta(1) subunit to the large conductance Ca(2+)-activated K(+) channel in the absence of Ca(2+). Increased Ca(2+) sensitivity from a Ca(2+)-independent mechanism.

Nimigean C M CM Magleby K L KL

The Journal of general physiology 20000601 6

Coexpression of the beta(1) subunit with the alpha subunit (mSlo) of BK channels increases the apparent Ca(2+) sensitivity of the channel. This study investigates whether the mechanism underlying the increased Ca(2+) sensitivity requires Ca(2+), by comparing the gating in 0 Ca(2+)(i) of BK channels composed of alpha subunits to those composed of alpha+beta(1) subunits. The beta(1) subunit increased burst duration approximately 20-fold and the duration of gaps between bursts approximately 3-fold, ...[more]

PMID: 10828246

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Project description:BackgroundVascular smooth muscle cells (VSMCs) phenotype switching is very important during the pathogenesis and progression of vascular diseases. However, it is not well understood how normal VSMCs maintain the differentiated state. The large-conductance Ca2+-activated K+ (BKCa) channels are widely expressed in VSMCs and regulate vascular tone. Nevertheless, there is limited understanding of the role of the BKCa channel in modulation of the VSMC phenotype.Methods and resultsWe assessed BKCa channel expression levels in normal and injured carotid arteries from rats of the balloon-injury model. A strong decrease of BKCa-β1 was seen in the injured carotid arteries, accompanied by a parallel decrease of the VSMC contractile markers. BKCa-β1 in primary rat aortic VSMCs was decreased with the increase of passage numbers and the stimulation of platelet-derived growth factor (PDGF)-BB. Conversely, transforming growth factor β upregulated BKCa-β1. Meanwhile, the BKCa-β1 level was positively associated with the levels of VSMC contractile proteins. Intravenous injection of PDGF-BB induced downregulation of BKCa-β1 expression in the carotid arteries. Knockdown of BKCa-β1 favored VSMC dedifferentiation, characterized by altered morphology, abnormal actin fiber organization, decreased contractile proteins expression and reduced contractile ability. Furthermore, the resultant VSMC dedifferentiated phenotype rendered increased proliferation, migration, enhanced inflammatory factors levels, and matrix metalloproteinases activity. Studies using primary cultured aortic VSMCs from human recapitulated key findings. Finally, protein level of BKCa-β1 was reduced in human atherosclerotic arteries.ConclusionBKCa-β1 is important in the maintenance of the contractile phenotype of VSMCs. As a novel endogenous defender that prevents pathological VSMC phenotype switching, BKCa-β1 may serve as a potential therapeutic target for treating vascular diseases including post-injury restenosis and atherosclerosis.

Project description:The large conductance Ca(2+)-activated K(+) or BK channel has a role in sensory/neuronal excitation, intracellular signaling, and metabolism. In the non-mammalian cochlea, the onset of BK during development correlates with increased hearing sensitivity and underlies frequency tuning in non-mammals, whereas its role is less clear in mammalian hearing. To gain insights into BK function in mammals, coimmunoprecipitation and two-dimensional PAGE, combined with mass spectrometry, were used to reveal 174 putative BKAPs from cytoplasmic and membrane/cytoskeletal fractions of mouse cochlea. Eleven BKAPs were verified using reciprocal coimmunoprecipitation, including annexin, apolipoprotein, calmodulin, hippocalcin, and myelin P0, among others. These proteins were immunocolocalized with BK in sensory and neuronal cells. A bioinformatics approach was used to mine databases to reveal binary partners and the resultant protein network, as well as to determine previous ion channel affiliations, subcellular localization, and cellular processes. The search for binary partners using the IntAct molecular interaction database produced a putative global network of 160 nodes connected with 188 edges that contained 12 major hubs. Additional mining of databases revealed that more than 50% of primary BKAPs had prior affiliations with K(+) and Ca(2+) channels. Although a majority of BKAPs are found in either the cytoplasm or membrane and contribute to cellular processes that primarily involve metabolism (30.5%) and trafficking/scaffolding (23.6%), at least 20% are mitochondrial-related. Among the BKAPs are chaperonins such as calreticulin, GRP78, and HSP60 that, when reduced with siRNAs, alter BKalpha expression in CHO cells. Studies of BKalpha in mitochondria revealed compartmentalization in sensory cells, whereas heterologous expression of a BK-DEC splice variant cloned from cochlea revealed a BK mitochondrial candidate. The studies described herein provide insights into BK-related functions that include not only cell excitation, but also cell signaling and apoptosis, and involve proteins concerned with Ca(2+) regulation, structure, and hearing loss.

Project description:1. Nine bis-quinolinyl and bis-quinolinium compounds related to dequalinium, and previously shown to block apamin-sensitive small conductance Ca(2+)-activated K(+) channels (SK(Ca)), have been tested for their inhibitory effects on actions mediated by intermediate conductance Ca(2+)-activated K(+) channels (IK(Ca)) in rabbit blood cells. 2. In most experiments, a K(+)-sensitive electrode was employed to monitor the IK(Ca)-mediated net loss of cell K(+) that followed the addition of the Ca(2+) ionophore A23187 (2 microM) to red cells suspended at an haematocrit of 1% in a low K(+) (0.12 - 0.17 mM) solution. The remainder used an optical method based on measuring the reduction in light transmission that occurred on applying A23187 (0.4 or 2 microM) to a very dilute suspension of red cells (haematocrit 0.02%). 3. Of the compounds tested, the most potent IK(Ca) blocker was 1,12 bis[(2-methylquinolin-4-yl)amino]dodecane (UCL 1407) which had an IC(50) of 0.85+/-0.06 microM (mean+/-s.d. mean). 4. The inhibitory action of UCL 1407 and its three most active congeners was characterized by (i) a Hill slope greater than unity, (ii) sensitivity to an increase in external [K(+)], and (iii) a time course of onset that suggested use-dependence. Also, the potency of the nonquaternary compounds tested increased with their predicted lipophilicity. These findings suggested that the IK(Ca) blocking action resembles that of cetiedil rather than of clotrimazole. 5. Some quaternized members of the series were also active. The most potent was the monoquaternary UCL 1440 ((1-[N-[1-(3, 5-dimethoxybenzyl)-2-methylquinolinium-4-yl]amino]-10-[N'-(2-me thylqu inolinium-4yl)amino] decane (trifluoroacetate) which had an IC(50) of 1.8+/-0.1 microM. The corresponding bisquaternary UCL 1438 (1, 10-bis[N-[1-(3,5-dimethoxybenzyl)-2-methylquinolinium-4-yl]amino] decane bis(trifluoroacetate) was almost as active (IC(50) 2.7+/-0.3 microM). 6. A bis-aminoquinolium cyclophane (UCL 1684) had little IK(Ca) blocking action despite its great potency at SK(Ca) channels (IC(50) 4.1+/-0.2 nM). 7. The main outcome is the identification of new intermediate-conductance Ca(2+)-activated K(+) channel blockers with a wide range of IK(Ca)/SK(Ca) selectivities.

Dataset Information

Functional coupling of the beta(1) subunit to the large conductance Ca(2+)-activated K(+) channel in the absence of Ca(2+). Increased Ca(2+) sensitivity from a Ca(2+)-independent mechanism.

Publications

Functional coupling of the beta(1) subunit to the large conductance Ca(2+)-activated K(+) channel in the absence of Ca(2+). Increased Ca(2+) sensitivity from a Ca(2+)-independent mechanism.

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