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Background: Enzymes controlling (de)palmitoylation of ion channels are poorly defined. Results: Palmitoylation of BK channels by zDHHC22 and zDHHC23 and depalmitoylation by LYPLA1 and LYPLAL1 controls BK channel cell surface expression. Conclusion: Acyl protein transferases and thioesterases display substrate specificity and control BK channel surface expression. Significance: Understanding how channels are (de)palmitoylated is essential for defining the role of palmitoylation in ion channel physiology., Protein palmitoylation is rapidly emerging as an important determinant in the regulation of ion channels, including large conductance calcium-activated potassium (BK) channels. However, the enzymes that control channel palmitoylation are largely unknown. Indeed, although palmitoylation is the only reversible lipid modification of proteins, acyl thioesterases that control ion channel depalmitoylation have not been identified. Here, we demonstrate that palmitoylation of the intracellular S0–S1 loop of BK channels is controlled by two of the 23 mammalian palmitoyl-transferases, zDHHC22 and zDHHC23. Palmitoylation by these acyl transferases is essential for efficient cell surface expression of BK channels. In contrast, depalmitoylation is controlled by the cytosolic thioesterase APT1 (LYPLA1), but not APT2 (LYPLA2). In addition, we identify a splice variant of LYPLAL1, a homolog with ∼30% identity to APT1, that also controls BK channel depalmitoylation. Thus, both palmitoyl acyltransferases and acyl thioesterases display discrete substrate specificity for BK channels. Because depalmitoylated BK channels are retarded in the trans-Golgi network, reversible protein palmitoylation provides a critical checkpoint to regulate exit from the trans-Golgi network and thus control BK channel cell surface expression.

In the hippocampus, BK channels are preferentially localized in presynaptic glutamatergic terminals including mossy fibers where they are thought to play an important role regulating excessive glutamate release during hyperactive states. Large conductance calcium-activated potassium channels (BK, MaxiK, Slo) have recently been implicated in the pathogenesis of genetic epilepsy. However, the role of BK channels in acquired mesial temporal lobe epilepsy (MTLE) remains unknown. Here we used immunohistochemistry, laser scanning confocal microscopy (LSCM), Western immunoblotting and RT-PCR to investigate the expression pattern of the alpha-pore-forming subunit of BK channels in the hippocampus and cortex of chronically epileptic rats obtained by the pilocarpine model of MTLE. All epileptic rats experiencing recurrent spontaneous seizures exhibited a significant down-regulation of BK channel immunostaining in the mossy fibers at the hilus and stratum lucidum of the CA3 area. Quantitative analysis of immunofluorescence signals by LSCM revealed a significant 47% reduction in BK channel immunofluorescent signals in epileptic rats when compared to age-matched non-epileptic control rats. These data correlate with a similar reduction in BK channel protein levels and transcripts in the cortex and hippocampus. Our data indicate a seizure-related down-regulation of BK channels in chronically epileptic rats. Further functional assays are necessary to determine whether altered BK channel expression is an acquired channelopathy or a compensatory mechanism affecting the network excitability in MTLE. Moreover, seizure-mediated BK down-regulation may disturb neuronal excitability and presynaptic control at glutamatergic terminals triggering exaggerated glutamate release and seizures.

Small-conductance, Ca2+ activated K+ channels (SK channels) are expressed at high levels in brain regions responsible for learning and memory. In the current study we characterized the contribution of SK2 channels to synaptic plasticity and to different phases of hippocampal memory formation. Selective SK2 antisense-treatment facilitated basal synaptic transmission and theta-burst induced LTP in hippocampal brain slices. Using the selective SK2 antagonist Lei-Dab7 or SK2 antisense probes, we found that hippocampal SK2 channels are critical during two different time windows: 1) blockade of SK2 channels before the training impaired fear memory, whereas, 2) blockade of SK2 channels immediately after the training enhanced contextual fear memory. We provided the evidence that the post-training cleavage of the SK2 channels was responsible for the observed bidirectional effect of SK2 channel blockade on memory consolidation. Thus, Lei-Dab7-injection before training impaired the C-terminal cleavage of SK2 channels, while Lei-Dab7 given immediately after training facilitated the C-terminal cleavage. Application of the synthetic peptide comprising a leucine-zipper domain of the C-terminal fragment to Jurkat cells impaired SK2 channel-mediated currents, indicating that the endogenously cleaved fragment might exert its effects on memory formation by blocking SK2 channel-mediated currents. Our present findings suggest that SK2 channel proteins contribute to synaptic plasticity and memory not only as ion channels but also by additionally generating a SK2 C-terminal fragment, involved in both processes. The modulation of fear memory by down-regulating SK2 C-terminal cleavage might have applicability in the treatment of anxiety disorders in which fear conditioning is enhanced.

Autoreactive memory T lymphocytes are implicated in the pathogenesis of autoimmune diseases. Here we demonstrate that disease-associated autoreactive T cells from patients with type-1 diabetes mellitus or rheumatoid arthritis (RA) are mainly CD4+CCR7−CD45RA−effector memory T cells (TEMcells) with elevated Kv1.3 potassium channel expression. In contrast, T cells with other antigen specificities from these patients, or autoreactive T cells from healthy individuals and disease controls, express low levels of Kv1.3 and are predominantly naïve or central-memory (TCM) cells. In TEMcells, Kv1.3 traffics to the immunological synapse during antigen presentation where it colocalizes with Kvβ2, SAP97, ZIP, p56lck, and CD4. Although Kv1.3 inhibitors [ShK(L5)-amide (SL5) and PAP1] do not prevent immunological synapse formation, they suppress Ca2+-signaling, cytokine production, and proliferation of autoantigen-specific TEMcells at pharmacologically relevant concentrations while sparing other classes of T cells. Kv1.3 inhibitors ameliorate pristane-induced arthritis in rats and reduce the incidence of experimental autoimmune diabetes in diabetes-prone (DP-BB/W) rats. Repeated dosing with Kv1.3 inhibitors in rats has not revealed systemic toxicity. Further development of Kv1.3 blockers for autoimmune disease therapy is warranted.

The role of ion channels in cell physiology is regulated by processes occurring after protein biosynthesis, which are critical for both channel function and targeting of channels to appropriate cell compartments. Here we apply biochemical and electrophysiological methods to investigate the role of the high-conductance, calcium-activated potassium (Maxi-K) channel C-terminal domain in channel tetramerization, association with the beta1 subunit, trafficking of the channel complex to the cell surface, and channel function. No evidence for channel tetramerization, cell surface expression, or function was observed with Maxi-K(1)(-)(323), a construct truncated three residues after the S(6) transmembrane domain. However, Maxi-K(1)(-)(343) and Maxi-K(1)(-)(441) are able to form tetramers and to associate with the beta1 subunit. Maxi-K(1)(-)(343)-beta1 and Maxi-K(1)(-)(441)-beta1 complexes are efficiently targeted to the cell surface and cannot be pharmacologically distinguished from full-length channels in binding experiments but do not form functional channels. Maxi-K(1)(-)(651) forms tetramers and associates with beta1; however, the complex is not present at the cell surface, but is retained intracellularly. Maxi-K(1)(-)(651) surface expression and channel function can be fully rescued after coexpression with its C-terminal complement, Maxi-K(652)(-)(1113). However coexpression of Maxi-K(1)(-)(343) and Maxi-K(1)(-)(441) with their respective C-terminal complements did not rescue channel function. Together, these data demonstrate that the domain(s) in the Maxi-K channel necessary for formation of tetramers, coassembly with the beta1 subunit, and cell surface expression resides within the S(0)-S(6) linker domain of the protein, and that structural constraints within the gating ring in the C-terminal region can regulate trafficking and function of constructs truncated in this region.

Large conductance voltage- and calcium-activated potassium (BK Ca ) channels are important signaling molecules that are regulated by multiple protein kinases and protein phosphatases at multiple sites. The pore-forming α-subunits, derived from a single gene that undergoes extensive alternative pre-mRNA splicing, assemble as tetramers. Although consensus phosphorylation sites have been identified within the C-terminal domain of α-subunits, it is not known whether phosphorylation of all or single α-subunits within the tetramer is required for functional regulation of the channel. Here, we have exploited a strategy to study single-ion channels in which both the α-subunit splice-variant composition is defined and the number of consensus phosphorylation sites available within each tetramer is known. We have used this approach to demonstrate that cAMP-dependent protein kinase (PKA) phosphorylation of the conserved C-terminal PKA consensus site (S899) in all four α-subunits is required for channel activation. In contrast, inhibition of BK Ca channel activity requires phosphorylation of only a single α-subunit at a splice insert (STREX)-specific PKA consensus site (S4 STREX ). Thus, distinct modes of BK Ca channel regulation by PKA phosphorylation exist: an “all-or-nothing” rule for activation and a “single-subunit” rule for inhibition. This essentially digital regulation has important implications for the combinatorial and conditional regulation of BK Ca channels by reversible protein phosphorylation.

Adrenal glucocorticoids exert powerful effects on cellular excitability in neuroendocrine cells and neurons, although the underlying mechanisms are poorly understood. In metabolically intact mouse anterior pituitary corticotrope (AtT20) cells glucocorticoid-induced proteins render large conductance calcium-activated potassium (BK) channels insensitive to inhibition by protein kinase A (PKA). In this study we have addressed whether this action of glucocorticoids is mediated via protein phosphatase activity at the level of single BK channels. In isolated inside-out patches from control AtT20 cells BK channels (125 pS) were inhibited by activation of closely associated PKA. Pretreatment (2 h) of cells with 1 μm dexamethasone before patch excision did not modify the intrinsic properties or expression levels of BK channel α-subunits in AtT20 cells. However, PKA-mediated inhibition of BK channel activity in isolated patches from steroid-treated cells was severely blunted. This effect of steroid was not observed using adenosine 5′-O-(3-thiotriphosphate) as phosphate donor or on exposure of the intracellular face of the patch with 10 nm of the protein phosphatase inhibitors okadaic acid or calyculin A but was mimicked by application of protein phosphatase 2A (PP2A) to the intracellular face of patches from control cells. Glucocorticoids did not modify total PP2A activity in AtT20 cells, suggesting that modified PP2A-like phosphatase activity closely associated with BK channels is required for glucocorticoid action.

Non-technical summary Our ability to respond to stress is critically dependent upon the release of the stress hormone adrenocorticotrophic hormone (ACTH) from corticotroph cells of the anterior pituitary gland. ACTH release is controlled by the electrical properties of corticotrophs that are determined by the movement of ions through channel pores in the plasma membrane. We show that a calcium-activated potassium ion channel called SK4 is expressed in corticotrophs and regulates ACTH release. We provide evidence of how SK4 channels control corticotroph function, which is essential for understanding homeostasis and for treating stress-related disorders. Abstract The anterior pituitary corticotroph is a major control point for the regulation of the hypothalamic–pituitary–adrenal (HPA) axis and the neuroendocrine response to stress. Although corticotrophs are known to be electrically excitable, ion channels controlling the electrical properties of corticotrophs are poorly understood. Here, we exploited a lentiviral transduction system to allow the unequivocal identification of live murine corticotrophs in culture. We demonstrate that corticotrophs display highly heterogeneous spontaneous action-potential firing patterns and their resting membrane potential is modulated by a background sodium conductance. Physiological concentrations of corticotrophin-releasing hormone (CRH) and arginine vasopressin (AVP) cause a depolarization of corticotrophs, leading to a sustained increase in action potential firing. A major component of the outward potassium conductance was mediated via intermediate conductance calcium-activated (SK4) potassium channels. Inhibition of SK4 channels with TRAM-34 resulted in an increase in corticotroph excitability and exaggerated CRH/AVP-stimulated ACTH secretion in vitro. In accordance with a physiological role for SK4 channels in vivo, restraint stress-induced plasma ACTH and corticosterone concentrations were significantly enhanced in gene-targeted mice lacking SK4 channels (Kcnn4−/−). In addition, Kcnn4−/− mutant mice displayed enhanced hypothalamic c-fos and nur77 mRNA expression following restraint, suggesting increased neuronal activation. Thus, stress hyperresponsiveness observed in Kcnn4−/− mice results from enhanced secretagogue-induced ACTH output from anterior pituitary corticotrophs and may also involve increased hypothalamic drive, thereby suggesting an important role for SK4 channels in HPA axis function.

Trafficking of the pore-forming alpha-subunits of large conductance calcium-and voltage-activated potassium (BK) channels to the cell surface represents an important regulatory step in controlling BK channel function. Here, we identify multiple trafficking signals within the intracellular RCK1-RCK2 linker of the cytosolic C terminus of the channel that are required for efficient cell surface expression of the channel. In particular, an acidic cluster-like motif was essential for channel exit from the endoplasmic reticulum and subsequent cell surface expression. This motif could be transplanted onto a heterologous nonchannel protein to enhance cell surface expression by accelerating endoplasmic reticulum export. Importantly, we identified a human alternatively spliced BK channel variant, hSlo Delta(579-664), in which these trafficking signals are excluded because of in-frame exon skipping. The hSlo Delta(579-664) variant is expressed in multiple human tissues and cannot form functional channels at the cell surface even though it retains the putative RCK domains and down stream trafficking signals. Functionally, the hSlo Delta(579-664) variant acts as a dominant negative subunit to suppress cell surface expression of BK channels. Thus alternative splicing of the intracellular RCK1-RCK2 linker plays a critical role in determining cell surface expression of BK channels by controlling the inclusion/exclusion of multiple trafficking motifs.

Large conductance calcium- and voltage-gated potassium (BK) channels are important regulators of physiological homeostasis and their function is potently modulated by protein kinase A (PKA) phosphorylation. PKA regulates the channel through phosphorylation of residues within the intracellular C terminus of the pore-forming α-subunits. However, the molecular mechanism(s) by which phosphorylation of the α-subunit effects changes in channel activity are unknown. Inhibition of BK channels by PKA depends on phosphorylation of only a single α-subunit in the channel tetramer containing an alternatively spliced insert (STREX) suggesting that phosphorylation results in major conformational rearrangements of the C terminus. Here, we define the mechanism of PKA inhibition of BK channels and demonstrate that this regulation is conditional on the palmitoylation status of the channel. We show that the cytosolic C terminus of the STREX BK channel uniquely interacts with the plasma membrane via palmitoylation of evolutionarily conserved cysteine residues in the STREX insert. PKA phosphorylation of the serine residue immediately upstream of the conserved palmitoylated cysteine residues within STREX dissociates the C terminus from the plasma membrane, inhibiting STREX channel activity. Abolition of STREX palmitoylation by site-directed mutagenesis or pharmacological inhibition of palmitoyl transferases prevents PKA-mediated inhibition of BK channels. Thus, palmitoylation gates BK channel regulation by PKA phosphorylation. Palmitoylation and phosphorylation are both dynamically regulated; thus, cross-talk between these 2 major posttranslational signaling cascades provides a mechanism for conditional regulation of BK channels. Interplay of these distinct signaling cascades has important implications for the dynamic regulation of BK channels and physiological homeostasis.

Previous studies have shown that tumor necrosis factor-alpha (TNF-alpha) induces neuroprotection against excitotoxic damage in primary cortical neurons via sustained nuclear factor-kappa B (NF-kappa B) activation. The transcription factor NF-kappa B can regulate the expression of small conductance calcium-activated potassium (K(Ca)) channels. These channels reduce neuronal excitability and as such may yield neuroprotection against neuronal overstimulation. In the present study we investigated whether TNF-alpha-mediated neuroprotective signaling is inducing changes in the expression of small conductance K(Ca) channels. Interestingly, the expression of K(Ca)2.2 channel was up-regulated by TNF-alpha treatment in a time-dependent manner whereas the expression of K(Ca)2.1 and K(Ca)2.3 channels was not altered. The increase in K(Ca)2.2 channel expression after TNF-alpha treatment was shown to be dependent on TNF-R2 and NF-kappa B activation. Furthermore, activation of small conductance K(Ca) channels by 6,7-dichloro-1H-indole-2,3-dione 3-oxime or cyclohexyl-[2-(3,5-dimethyl-pyrazol-1-yl)-6-methyl-pyrimidin-4-yl]-amine-induced neuroprotection against a glutamate challenge. Treatment with the small conductance K(Ca) channel blocker apamin or K(Ca)2.2 channel siRNA reverted the neuroprotective effect elicited by TNF-alpha. We conclude that treatment of primary cortical neurons with TNF-alpha leads to increased K(Ca)2.2 channel expression which renders neurons more resistant to excitotoxic cell death.

Stress activates the hypothalamic-pituitary-adrenal (HPA) axis, releasing ACTH from the anterior pituitary gland and glucocorticoids from the adrenal cortex. Stress also activates the sympathetic nervous system, evoking adrenaline release from the adrenal medulla. Large-conductance calcium- and voltage-activated potassium (BK) channels have been implicated in regulation of cellular excitability in these systems. Here, we examine the functional role of BK channels in HPA axis regulation in vivo using female mice genetically deficient (BK / ) for the pore-forming subunits of BK channels. BK / phenotype in the HPA was confirmed by immunohistochemistry, Western blot analysis, and corticotrope patch-clamp recording. Restraint stress-induced plasma concentrations of ACTH and corticosterone were significantly blunted in BK / mice compared with wild type (WT) controls. This stress hyporesponsiveness was associated with reduced activation of hypothalamic paraventricular nucleus (PVN) neurons. Basal expression of CRH, but not arginine vasopressin mRNA in the PVN was significantly lower in BK / mice compared with WT controls. Total anterior pituitary ACTH peptide content, but not proopiomelanocortin mRNA expression or corticotrope number, was significantly reduced in BK / mice compared with WT. However, anterior pituitary corticotropes from BK / mice fully supported ACTH output, releasing a significantly greater proportion of stored ACTH in response to secretagogue in vitro compared with WT. These results support an important role for BK channels in both the neural circuitry and endocrine output of the HPA axis and indicate that the stress hyporesponsiveness in BK / mice primarily results from reduced activation of hypothalamic PVN neurosecretory neurons. (Endocrinology 148: 5496–5506, 2007)

Calcium-activated potassium (BK) channels play a central role in regulating multiple physiological processes, from the control of blood flow to neuronal excitability. Coordinated regulation of BK channel activity by changes in actin cytoskeleton dynamics has been implicated in several of these processes and related disease states such as epilepsy and stroke. However, how BK channels interact with the actin cytoskeleton is essentially unknown. Here we demonstrate noncanonical Src homology domain 3 (SH3) binding site motifs in the intracellular C terminus of the BK channel pore-forming alpha-subunit that are conserved from fish to humans. These noncanonical motifs target multiple SH3 domain cellular signaling proteins to BK channels, including the SH3 adapter protein cortactin (EMS1). We demonstrate that cortactin provides a molecular bridge between BK channels and the cortical actin cytoskeleton in cells. Disruption of the SH3-mediated interaction prevents the regulation of BK channel activity controlled by changes in actin cytoskeletal dynamics. Targeting of cortactin to BK channels via a novel, noncanonical SH3 domain binding motif has important implications for the coordination of BK channel function in normal physiology and disease.

Background Large conductance calcium- and voltage activated potassium (BK) channels are important determinants of neuronal excitability through effects on action potential duration, frequency and synaptic efficacy. The pore- forming subunits are encoded by a single gene, KCNMA1, which undergoes extensive alternative pre mRNA splicing. Different splice variants can confer distinct properties on BK channels. For example, insertion of the 58 amino acid stress-regulated exon (STREX) insert, that is conserved throughout vertebrate evolution, encodes channels with distinct calcium sensitivity and regulation by diverse signalling pathways compared to the insertless (ZERO) variant. Thus, expression of distinct splice variants may allow cells to differentially shape their electrical properties during development. However, whether differential splicing of BK channel variants occurs during development of the mammalian CNS has not been examined. Results Using quantitative real-time polymerase chain reaction (RT-PCR) Taqman™ assays, we demonstrate that total BK channel transcripts are up regulated throughout the murine CNS during embryonic and postnatal development with regional variation in transcript levels. This upregulation is associated with a decrease in STREX variant mRNA expression and an upregulation in ZERO variant expression. Conclusion As BK channel splice variants encode channels with distinct functional properties the switch in splicing from the STREX phenotype to ZERO phenotype during embryonic and postnatal CNS development may provide a mechanism to allow BK channels to control distinct functions at different times of mammalian brain development.

The pore-forming alpha-subunits of large conductance calcium- and voltage-activated potassium (BK) channels are encoded by a single gene that undergoes extensive alternative pre-mRNA splicing. However, the extent to which differential exon usage at a single site of splicing may confer functionally distinct properties on BK channels is largely unknown. Here we demonstrated that alternative splicing at site of splicing C2 in the mouse BK channel C terminus generates five distinct splice variants: ZERO, e20, e21(STREX), e22, and a novel variant deltae23. Splice variants display distinct patterns of tissue distribution with e21(STREX) expressed at the highest levels in adult endocrine tissues and e22 at embryonic stages of mouse development. deltae23 is not functionally expressed at the cell surface and acts as a dominant negative of cell surface expression by trapping other BK channel splice variant alpha-subunits in the endoplasmic reticulum and perinuclear compartments. Splice variants display a range of biophysical properties. e21(STREX) and e22 variants display a significant left shift (>20 mV at 1 microM [Ca2+]i) in half-maximal voltage of activation compared with ZERO and e20 as well as considerably slower rates of deactivation. Splice variants are differentially sensitive to phosphorylation by endogenous cAMP-dependent protein kinase; ZERO, e20, and e22 variants are all activated, whereas e21 (STREX) is the only variant that is inhibited. Thus alternative pre-mRNA splicing from a single site of splicing provides a mechanism to generate a physiologically diverse complement of BK channel alpha-subunits that differ dramatically in their tissue distribution, trafficking, and regulation.

K+ channels represent a large and diverse group of ion channels that play a fundamental role in controlling cell excitability. These channels regulate hormone release from endocrine cells, modulate the pattern of transmitter release from neurons, and set the level of contraction in arterial smooth muscle cells. At first glance, K+ channels can be subdivided into either voltage-gated K+ (Kv) channels or ligand-gated K+ channels, depending on the stimulus that triggers the conformational change that leads to channel opening. Within the wide family of K+ channels gated by voltage, the Kv channels are activated solely by membrane depolarization, whereas high-conductance Ca2+-activated K+ (BKCa) channels require both membrane depolarization and an increased level of cytosolic free Ca2+ ([Ca2+]i) to activate effectively. The use of molecular biology techniques has greatly extended our current understanding regarding the structure and existence of subfamilies of K+ channels. However, three major questions still are the subject of extensive investigation: (1) What is the molecular composition of the different types of K+ channels expressed in vivo, (2) what is the physiological function of these K+ channels, and (3) is the molecular composition and functional profile of K+ channels altered in pathophysiological states?

Abstract —The Ca 2+ -sensitive K + channel (K Ca channel) plays a key role in buffering pressure-induced constriction of small cerebral arteries. An amplified current through this channel has been reported in vascular smooth muscle cells obtained from hypertensive animals, implying that the expression or properties of K Ca channels may be regulated by in vivo blood pressure levels. In this study, we investigated this hypothesis and its functional relevance by comparing the properties, expression levels, and physiological role of K Ca channels in cerebral resistance arteries from normotensive and genetically hypertensive rats. Whole-cell patch-clamp experiments revealed a 4.7-fold higher density of iberiotoxin-sensitive K Ca channel current at physiological membrane potentials in spontaneously hypertensive rat (SHR) compared with Wistar-Kyoto (WKY) rat cerebrovascular smooth muscle cells (n=18 and 21, respectively). However, additional single-channel analysis in detached patches showed similar levels of unitary conductance, voltage, and Ca 2+ sensitivity in K Ca channels from WKY and from SHR membranes. In contrast, Western analysis using an antibody directed against the K Ca channel α-subunit revealed a 4.1-fold increase in the corresponding 125-kD immunoreactive signal in cerebrovascular membranes from SHR compared with WKY rats. The functional impact of this enhanced K Ca channel expression was assessed in SHR and WKY rat pial arterioles, which were monitored by intravital microscopy through in situ cranial windows. Progressive pharmacological block of K Ca channels by iberiotoxin (0.1 to 100 nmol/L) dose-dependently constricted pial arterioles from SHR and WKY rats (n=6 to 8). The arterioles in SHR constricted 2- to 4-fold more intensely, and vasospasm occurred in some vessels. These data provide the first direct evidence that elevated levels of in situ blood pressure induce K Ca channel expression in cerebrovascular smooth muscle membranes. This homeostatic mechanism may critically regulate the resting tone of cerebral arterioles during chronic hypertension. Furthermore, the overexpression of distinct K + channel types during specific cardiovascular pathologies may provide for the upregulation of novel disease-specific membrane targets for vasodilator therapies.

Abstract Potassium efflux through Ca 2+ -sensitive K + channels (K Ca channels) is increased in arterial smooth muscle cells from hypertensive rats, but the molecular mechanism is unknown. The goal of this study was to compare the levels of K Ca channel current between aortic smooth muscle cells from adult Wistar-Kyoto rats (WKY) and spontaneously hypertensive rats (SHR) and then use Western blot methods and ribonuclease protection assays to examine the expression and mRNA levels for the K Ca channel in these same vascular tissues. Whole-cell patch-clamp methods indicated a larger component of K Ca channel current, sensitive to block by iberiotoxin (100 nmol/L), in single aortic smooth muscle cells from SHR compared with WKY. Subsequent Western blot analysis using a site-specific antibody (anti–α 913–926 ) directed against the S9/S10 linker of the α-subunit of the K Ca channel revealed a 125-kD immunoreactive band in lanes loaded with either WKY or SHR aortic muscle membranes. The immunoreactive density of this band, which corresponded to the known molecular size of the α-subunit, was 2.2-fold greater in lanes loaded with aortic smooth muscle membranes from the hypertensive animals. However, despite this evidence for an increased expression and functional enhancement of K Ca channels in aortic smooth muscle membranes of SHR, ribonuclease protection assays with a 32 P-labeled riboprobe targeted against the S9/S10 linker of the K Ca channel α-subunit revealed no difference in mRNA levels for the α-subunit between WKY and SHR aortic tissue. These findings provide initial evidence that (1) an increased expression of K Ca channels may be a mechanism for the enhanced K Ca current in aortic smooth muscle membranes of SHR, and (2) the upregulation of K Ca channels in arterial muscle membranes during hypertension, which is regarded as a homeostatic mechanism for buffering vascular excitability, may rely on post-transcriptional events.

Monoiodotyrosine margatoxin ([125I]MgTX) specifically and reversibly labels a maximum of 0.8 pmol of sites/mg of protein in purified rat brain synaptic plasma membrane vesicles with a dissociation constant of 0.1 pM under equilibrium binding conditions. This Kd value was confirmed by kinetic experiments (Kd of 0.07 pM), competition assays employing native margatoxin (MgTX) (Ki of 0.15 pM), and receptor saturation studies (Kd of 0.18 pM). Thus, this toxin represents the highest affinity, reversible radioligand for any membrane-bound receptor or ion channel described to date. [125I]MgTX binding in this system is modulated by charybdotoxin (Ki of 5 pM), kaliotoxin (Ki of 1.5 pM), and the agitoxins I and II (Ki's of 0.1 and 0.3 pM, respectively), in a noncompetitive manner. Moreover, alpha-dendrotoxin displayed a Ki value of 0.5 pM. Iberiotoxin was without any effect, suggesting that the receptor site is likely to be associated with a voltage-gated K+ channel complex. [125I]MgTX binding is inhibited by cations that are established blockers of voltage-dependent K+ channels (Ba2+, Ca2+, Cs+). The monovalent cations Na+ and K+ stimulate binding at low concentrations before producing complete inhibition as their concentrations are increased. Stimulation of binding results from an allosteric interaction that decreases Kd, whereas inhibition is due to an ionic strength effect. Affinity labeling of the binding site in rat brain synaptic plasma membranes employing [125I]MgTX and the bifunctional cross-linking reagent, disuccinimidyl suberate, causes specific and covalent incorporation of toxin into a glycoprotein of an apparent molecular weight (M(r)) of 74,000. Deglycosylation studies reveal an M(r) for the core polypeptide of the MgTX receptor of 63,000.(ABSTRACT TRUNCATED AT 250 WORDS)

High-conductance calcium-activated potassium (maxi-K) channels are composed of two subunits, alpha and beta. The pore-forming alpha subunit is a member of the mSlo family of K+ channels, whereas the beta subunit is a novel protein that modulates the biophysical and pharmacological properties of the channel complex. In the presence of a bifunctional cross-linking reagent, monoiodotyrosine charybdotoxin ([125I]ChTX) is covalently incorporated specifically into Lys69 of the beta subunit, which is located in a large extracellular loop of this protein. Using variants of ChTX which retain their channel-blocking activity and in which individual Lys residues have been mutated, we have identified the corresponding amino acid in ChTX that is involved in the cross-linking reaction. All of the ChTX mutants investigated bind to the maxi-K channel and display the same pharmacological profile as native ChTX in competition binding experiments. Whereas substitution of amino acids at positions 11 and 31 of ChTX yields wild-type cross-linking patterns, the peptide without a Lys at position 32 fails to incorporate into the beta subunit of the maxi-K channel. Given the model for the interaction between ChTX and the outer vestibule of the maxi-K channel that has been proposed (Stampe et al., 1994), our data constrain the maximum distance between the pore of this channel and the region in the extracellular loop of the beta subunit where the cross-linking reaction takes place to 11 A. This topological limit helps define structural features of the maxi-K channel that may aide in probing the functional interaction between alpha and beta subunits of the channel complex.