Article

Conditional and unconditional inhibition of calcium-activated potassium channels by reversible protein phosphorylation

Laboratory of Signal Transduction, NIEHS, National Institutes of Health, Research Triangle Park, North Carolina 27709, USA.
Journal of Biological Chemistry (Impact Factor: 4.6). 03/2000; 275(6):3749-54. DOI: 10.1074/jbc.275.6.3749
Source: PubMed

ABSTRACT Large conductance, calcium-activated potassium channels (BK(Ca) or maxi-K) are important determinants of membrane excitability in many cell types. We used patch clamp techniques to study the biochemical regulation of native BK(Ca) channel proteins by endogenous Ser/Thr-directed protein kinases and phosphatases in cell-free membrane patches from rat pituitary tumor cells (GH(4)C(1)). When protein kinase activity was blocked by removing ATP, endogenous protein phosphatases slowly increased BK(Ca) channel activity approximately 3-fold. Dephosphorylated channels could be activated fully by physiological increases in cytoplasmic calcium or membrane depolarization. In contrast, endogenous protein kinases inhibited BK(Ca) channel activity at two functionally distinct sites. A closely associated, cAMP-dependent protein kinase rapidly reduced channel activity in a conditional manner that could be overcome completely by increasing cytoplasmic free calcium 3-fold or 20 mV further depolarization. Phosphorylation at a pharmacologically distinct site inhibited channel activity unconditionally by reducing availability to approximately half that of maximum at all physiological calcium and voltages. Conditional versus unconditional inhibition of BK(Ca) channel activity through different protein kinases provides cells with a powerful computational mechanism for regulating membrane excitability.

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    • "Patch clamp recordings revealed that STREX causes BK channels to activate at more negative potentials and enhances activation and decreases deactivation which leads to increased repetitive firing of action potentials. STREX can be artificially induced by growing cells in phenol red which causes a significant increase in channel sensitivity to inhibition by oxidation but also to Ca 2+ (Hall & Armstrong 2000). Coassembly of STREX/β1-subunits, however, could only be stimulated with a truncated Nterminus variation present which has physiological impact of channel regulation by Ca 2+ , oxidation, and phosphorylation. "
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    • "Patch clamp recordings revealed that STREX causes BK channels to activate at more negative potentials and enhances activation and decreases deactivation which leads to increased repetitive firing of action potentials. STREX can be artificially induced by growing cells in phenol red which causes a significant increase in channel sensitivity to inhibition by oxidation but also to Ca 2+ (Hall & Armstrong 2000). Coassembly of STREX/β1-subunits, however, could only be stimulated with a truncated Nterminus variation present which has physiological impact of channel regulation by Ca 2+ , oxidation, and phosphorylation. "
    Patch Clamp Technique, 03/2012; , ISBN: 978-953-51-0406-3
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    • "One molecular mechanism that may determine the cell-specific function of maxi-K channels is alternative splicing of the transcript encoding the pore-forming α-subunit (Saito et al. 1997). Splicing results in maxi-K channel isoforms that are differentially regulated by Ca 2+ and voltage, oestrogen, ACTH and post-translational modification by kinases (Dopico et al. 1994; Nara et al. 1998; Xie & McCobb, 1998; Hall & Armstrong, 2000; Tian et al. 2001; Zarei et al. 2001; Holdiman et al. 2002). Our laboratory previously identified and functionally characterized a human maxi-K channel α-subunit isoform (mK44) generated by introduction of 44 additional amino acids into the first intracellular loop of the canonical channel (Korovkina et al. 2001). "
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    ABSTRACT: Large conductance Ca(2+)- and voltage-activated K+ (maxi-K) channels modulate human myometrial smooth muscle cell (hMSMC) excitability; however, the role of individual alternatively spliced isoforms remains unclear. We have previously shown that the transcript of a human maxi-K channel isoform (mK44) is expressed predominantly in myometrial and aortic smooth muscle and forms a functional channel in heterologous expression systems. The mK44 isoform contains unique consensus motifs for both endoproteolytic cleavage and N-myristoylation, although the function of these post-translational modifications is unknown. The goal of these studies was to determine the role of post-translational modifications in regulating mK44 channel function in hMSMCs. An mK44-specific antibody indicated that this channel is localized intracellularly in hMSMCs and translocates to the cell membrane in response to increases in intracellular Ca(2+). Immunological analyses using an N-terminally myc-tagged mK44 construct demonstrated endoproteolytical cleavage of mK44 in hMSMCs resulting in membrane localization of the mK44 N-termini and intracellular retention of the pore-forming C-termini. Caffeine-induced Ca(2+) release from intracellular stores resulted in translocation of the C-termini of mK44 to the cell membrane and co-localization with its N-termini. Translocation of mK44 channels to the cell membrane was concomitant with repolarization of the hMSMCs. Endoproteolytic digest of mK44 did not occur in HEK293 cells or mouse fibroblasts. MK44 truncated at a putative N-myristoylation site did not produce current when expressed alone, but formed a functional channel when co-expressed with the N-terminus. These findings provide novel insight into cell-specific regulation of maxi-K channel function.
    The Journal of Physiology 07/2006; 573(Pt 2):329-41. DOI:10.1113/jphysiol.2006.106922 · 4.54 Impact Factor

David L. Armstrong