Iain Rowe

Palmitoylation gates phosphorylation-dependent regulation of BK potassium channels

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.

Characterization of an ATP‐modulated large conductance Ca(2+)‐activated K+ channel present in rat cortical neurones.

1. Single channel current recordings were used to study the characteristics of a large conductance Ca(2+)‐activated K+ (BKCa) channel present in neurones acutely dissociated from the rat motor cortex. Application of ATP to the intracellular surface of excised inside‐out patches produced a large, concentration‐dependent increase in BKCa channel activity. 2. This ATP‐mediated activation was dependent upon the presence of Mg2+ in the intracellular bathing solution and was diminished by the phosphatases 2,3‐butanedione monoxime (BDM) or alkaline phosphatase and by the protein kinase inhibitors staurosporine, H‐7 and PKI. 3. ADP stimulated BKCa channel activity in a Mg(2+)‐dependent manner, an action also inhibited by the concomitant application of PKI or BDM. The effect of ADP was reduced by application of hexokinase and glucose or by application of the adenylate kinase inhibitor Ap5A. 4. Of other nucleotides tested, only CTP consistently activated BKCa channel activity. 5. Using the cell‐attached configuration, bath application of forskolin or dibutyryl cAMP stimulated BKCa channel activity. 6. It is concluded that BKCa channel activity in the rat motor cortex is subject to modulation by the activity of a closely associated kinase. The ability of cAMP activators to stimulate BKCa channel activity in the intact cell suggests that this system may be of physiological importance.

Conditional protein phosphorylation regulates BK channel activity in rat cerebellar Purkinje neurons

Large conductance calcium‐ and voltage‐activated potassium (BK) channels are widely expressed in the mammalian central nervous system. Although the activity of BK channels in endocrine and vascular cells is regulated by protein kinases and phosphatases associated with the channel complex, direct evidence for such modulation in neurons is largely lacking. Single‐channel analysis from inside‐out patches isolated from the soma of dissociated rat cerebellar Purkinje neurons demonstrated that the activity of BK channels is regulated by multiple endogenous protein kinases and protein phosphatases in the membrane patch. The majority of BK channels were non‐inactivating and displayed a ‘low’ activity phenotype determined at +40 mV and 1 μM intracellular free calcium. These channels were activated by cAMP‐dependent protein kinase (PKA) associated with the patch and the extent of PKA activation was limited by an opposing endogenous type 2A‐like protein phosphatase (PP2A). Importantly, PKA activation was dependent upon the prior phosphorylation status of the BK channel complex dynamically controlled by protein kinase C (PKC) and protein phosphatase 1 (PP1). In contrast, Purkinje cells also displayed a low proportion of non‐inactivating BK channels with a ‘high’ activity under the same recording conditions and these channels were inhibited by endogenous PKA. Our data suggest that: (1) multiple endogenous protein kinases and phosphatases functionally couple to the BK channel complex to allow conditional modulation of BK channel activity in neurons, and (2) native, phenotypically distinct, neuronal BK channels are differentially sensitive to PKA‐dependent phosphorylation.

The high-affinity sulphonylurea receptor regulates KATP channels in nerve terminals of the rat motor cortex.

Abstract: The coexpression of sulphonylurea binding sites and ATP‐sensitive K+ (KATP) channels was examined in the rat motor cortex, an area of the CNS exhibiting a high density of sulphonylurea binding. These channels were not detected on neuronal cell bodies, but sulphonylurea‐sensitive KATP channels and charybdotoxin‐sensitive, large‐conductance calcium‐activated K+ BKCa channels were detected by patch clamping of fused nerve terminals from the motor cortex. Subcellular fractionation revealed that high‐affinity sulphonylurea binding sites were enriched in the nerve terminal fraction, whereas glibenclamide increased calcium‐independent glutamate efflux from isolated nerve terminals. It is concluded that neuronal sulphonylurea receptors and KATP channels are functionally linked in the motor cortex and that they are both selectively expressed in nerve terminals, where the KATP channel may serve to limit glutamate release under conditions of metabolic stress.

Selective expression in carotid body type I cells of a single splice variant of the large conductance calcium- and voltage-activated potassium channel confers regulation by AMP-activated protein kinase

Inhibition of large conductance calcium-activated potassium (BKCa) channels mediates, in part, oxygen sensing by carotid body type I cells. However, BKCa channels remain active in cells that do not serve to monitor oxygen supply. Using a novel, bacterially derived AMP-activated protein kinase (AMPK), we show that AMPK phosphorylates and inhibits BKCa channels in a splice variant-specific manner. Inclusion of the stress-regulated exon within BKCa channel α subunits increased the stoichiometry of phosphorylation by AMPK when compared with channels lacking this exon. Surprisingly, however, the increased phosphorylation conferred by the stress-regulated exon abolished BKCa channel inhibition by AMPK. Point mutation of a single serine (Ser-657) within this exon reduced channel phosphorylation and restored channel inhibition by AMPK. Significantly, RT-PCR showed that rat carotid body type I cells express only the variant of BKCa that lacks the stress-regulated exon, and intracellular dialysis of bacterially expressed AMPK markedly attenuated BKCa currents in these cells. Conditional regulation of BKCa channel splice variants by AMPK may therefore determine the response of carotid body type I cells to hypoxia.

Palmitoylation of the S0-S1 linker regulates cell surface expression of voltage- and calcium- activated potassium (BK) channels

S-Palmitoylation is rapidly emerging as an important post-translational mechanism to regulate ion channels. We have previously demonstrated that large conductance calcium- and voltage-activated potassium (BK) channels are palmitoylated within an alternatively spliced (STREX) insert. However, these studies also revealed that additional site(s) for palmitoylation must exist outside of the STREX insert, although the identity or the functional significance of these palmitoylated cysteine residues are unknown. Here, we demonstrate that BK channels are palmitoylated at a cluster of evolutionary conserved cysteine residues (Cys-53, Cys-54, and Cys-56) within the intracellular linker between the S0 and S1 transmembrane domains. Mutation of Cys-53, Cys-54, and Cys-56 completely abolished palmitoylation of BK channels lacking the STREX insert (ZERO variant). Palmitoylation allows the S0-S1 linker to associate with the plasma membrane but has no effect on single channel conductance or the calcium/voltage sensitivity. Rather, S0-S1 linker palmitoylation is a critical determinant of cell surface expression of BK channels, as steady state surface expression levels are reduced by ∼55% in the C53:54:56A mutant. STREX variant channels that could not be palmitoylated in the S0-S1 linker also displayed significantly reduced cell surface expression even though STREX insert palmitoylation was unaffected. Thus our work reveals the functional independence of two distinct palmitoylation-dependent membrane interaction domains within the same channel protein and demonstrates the critical role of S0-S1 linker palmitoylation in the control of BK channel cell surface expression.

Potassium channel dysfunction in hypothalamic glucose‐receptive neurones of obese Zucker rats.

1. We have shown, using intracellular and cell‐attached recordings, that glucose‐receptive (GR) neurones of obese Zucker rats exhibit abnormal electrophysiological responses to changes in extracellular glucose concentration, whereas GR neurones of lean Zucker and control rats respond normally. 2. In inside‐out recordings from obese rat GR neurones it was shown that the 150 pS ATP‐sensitive K+ (KATP) and the 160 pS calcium‐activated K+ (KCa) channels were absent, whereas both were present in GR neurones of lean Zucker and control rats. 3. The potassium channel most frequently observed in inside‐out patches from obese GR neurones was characterized by a conductance of 213 pS, was activated by raising internal calcium and inhibited by application of internal ATP. This channel (which we have termed Kfa) was not observed in lean or control rat GR neurones. 4. Tolbutamide (100 microM) was found to induce no effect or to elicit a small depolarization of obese rat GR neurones in the absence of glucose, in contrast to its clear excitatory actions on control or lean Zucker GR neurones. 5. Intracellular, cell‐attached and inside‐out recordings from obese rat non‐GR neurones showed that there was no alteration in their membrane properties or firing characteristics or in the characteristics of the large‐conductance calcium‐activated K+ channel (KCa) present in these neurones as compared with lean and control rats. 6. It is concluded that the Kfa channel is specific to GR neurones of obese Zucker rats and that the presence of this channel coupled with the absence of KATP and KCa channels results in the abnormal glucose‐sensing response of these neurones.

Activation by intracellular ATP of a potassium channel in neurones from rat basomedial hypothalamus.

1. Cell‐attached recordings from isolated glucose‐sensitive hypothalamic neurones show that on removal of extracellular glucose there is an increased action current frequency concomitant with decreased single‐channel activity. Conversely activation of single K+ channels was observed when extracellular glucose was increased. Isolation of membrane patches into the inside‐out configuration following cell‐attached recording demonstrated the presence of an ATP‐activated K+ channel. 2. The ATP‐activated K+ channel was characterized by a mean single‐channel conductance of 132 pS in symmetrical 140 mM KCl solutions. Single‐channel open‐state probability (Po) was not calcium dependent, and the presence of calcium did not prevent activation of the channel by ATP. 3. Activation of the channel by ATP was concentration dependent and the Po of the ATP‐activated channel was unaffected by membrane voltage, regardless of the degree of activation elicited by ATP. 4. Open and closed time histograms were constructed from inside‐out and cell‐attached recordings and were consistent with a single open and two closed states. Channel openings were grouped in bursts. Application of ATP, in isolated patches, and glucose, in cell‐attached patches, increased the burst duration and number of bursts per second and decreased the slow closed‐state time constant. In neither case was there a significant change in the fast closed‐state time constant nor the open‐state time constant. 5. The non‐hydrolysable ATP analogue adenylylimidodiphosphate (AMP(PNP)) and ‘Mg2(+)‐free’ ATP produced little change in the Po of the ATP‐activated K+ channel when applied to the intracellular surface of excised patches. These results suggest that activation of this channel is via an enzymic mechanism. 6. ADP, GTP and GDP also activated the channel in a Mg(2+)‐dependent manner. ADP and ATP activated the channel in an additive manner and neither GTP nor GDP inhibited channel activity induced by ATP. 7. It is concluded that the ATP‐activated K+ channel observed in isolated inside‐out patches from hypothalamic neurones is the same as the channel activated by an increase in the concentration of extracellular glucose in cell‐attached recordings from glucose‐sensitive neurones.

Direct demonstration of sulphonylurea‐sensitive KATP channels on nerve terminals of the rat motor cortex

We examined whether ATP‐sensitive potassium (KATP) channels are present on presynaptic terminals of the rat motor cortex, an area of the CNS exhibiting a high density of sulphonylurea binding. A novel fused nerve terminal preparation was developed which produced structures amenable to patch clamp methods. In inside‐out recordings a K+channel was observed which possessed all the major features of the Type 1 KATP channel, including sensitivity to ATP and the antidiabetic sulphonylureas.

Membrane Trafficking of Large Conductance Calcium-activated Potassium Channels Is Regulated by Alternative Splicing of a Transplantable, Acidic Trafficking Motif in the RCK1-RCK2 Linker

Trafficking of the pore-forming α-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, hSloΔ579–664, in which these trafficking signals are excluded because of in-frame exon skipping. The hSloΔ579–664 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 downstream trafficking signals. Functionally, the hSloΔ579–664 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.