Stimulation Of Channel Activity Research Articles

Untangling the inseparable life of an ABC transporter and a potassium channel: Towards understanding the structure-function relationship in the KATP channel complex.

KATP channels are unique protein complexes of a potassium channel, Kir6.1 or Kir6.2, and an ABC transporter, SUR1, SUR2A or SUR2B (splice variants of SUR2), in a 4:4 stoichiometry. Broadly expressed in many cell types and tissues, KATP channels couple changes in cellular metabolic activity with membrane excitability to control a wide range of physiological processes, including hormone secretion, control of vascular tone, learning and memory, and cardiac and neuronal protection against ischemic insults. In pancreatic β-cells KATP channels formed by Kir6.2 and SUR1 control insulin secretion, and gain- or loss-of-function mutations in these channels cause neonatal diabetes or congenital hyperinsulinism, respectively. Physiological activity of pancreatic KATP channels is regulated by intracellular ATP and ADP. Non-hydrolytic binding of ATP to Kir6.2 inhibits channel activity, while MgATP and MgADP binding to the nucleotide binding domains (NBDs) of SUR1 stimulates channel activity. Besides ATP and ADP, membrane phospholipids in particular PIP2 interacts with Kir6.2 and stimulates channel activity. In addition, pharmacological inhibitors and activators act on SUR1 to modulate channel activity. Although Kir6.2 and SUR1 harbor the primary binding sites for specific physiological and pharmacological ligands, both subunits participate in ligand regulation. How do the potassium channel and the silent ABC transporter work together to decipher signals from different ligands for gating has long been a major question in the KATP channel field. Here, I present structural and functional evidence that SUR1 contributes directly to ATP and PIP2 binding via its TMD0 and L0 linker to enhance Kir6.2 sensitivity to both ligands. Moreover, SUR1 regulates Kir6.2 activity allosterically via its dynamic interaction with the N-terminus of Kir6.2.

The open structure of a neonatal diabetes-causing variant of the pancreatic KATP channel determined using cryo-electron microscopy.

Pancreatic ATP-sensitive potassium (KATP) channels, comprising four Kir6.2 and four SUR1 subunits, control insulin secretion in β-cells by coupling glucose metabolism with membrane excitability. Regulation of KATP channels involves orchestrated interactions of their subunits, Kir6.2 and SUR1, and ligands. ATP and PIP2 are known to inhibit and stimulate channel activity, respectively. Although Kir6.2 harbors the primary binding sites for ATP and PIP2, SUR1 is required to confer high channel sensitivity to both ligands. Mutations in channel proteins that disrupt ATP and PIP2 gating cause insulin secretion disease. Kir6.2-Q52R is a neonatal diabetes mutation that results in channels with extremely high open probability and low ATP sensitivity. Previous studies suggest the Kir6.2-Q52R mutation stabilizes channel opening in a SUR1-dependent manner, but the structural mechanism is unknown. Here, we determined the cryoEM structure of the Kir6.2-Q52R4SUR14 channel in the absence of nucleotides or pharmacological ligands. This 3.3 Å resolution structure reveals that Kir6.2 is open, with its cytoplasmic domain (CTD) corkscrewed up to the membrane. The side-chain of Kir6.2-Q52R is flipped away from the ATP-binding site and embedded into SUR1-TMD0 in a cation-pi interaction with SUR1-W51 at the membrane. The cryo-EM map has clear density showing the first intracellular loop of SUR1-TMD0 (ICL1, 52-60) is ordered and forms main chain interactions between Kir6.2-βA (the CTD β-strand just N-terminal to the interfacial helix) and SUR1-ICL1, tethering the CTD to TMD0 to stabilize the open state. Our study provides structural insights into the mechanism by which the Kir6.2-Q52R mutation causes severe neonatal diabetes and how Kir6.2 and SUR1 interact to stabilize KATP channels in the open state.

Combining native mass spectrometry and lipidomics to uncover specific membrane protein-lipid interactions from natural lipid sources.

While it is known that lipids play an essential role in regulating membrane protein structure and function, it remains challenging to identify specific protein-lipid interactions. Here, we present an innovative approach that combines native mass spectrometry (MS) and lipidomics to identify lipids retained by membrane proteins from natural lipid extracts. Our results reveal that the bacterial ammonia channel (AmtB) enriches specific cardiolipin (CDL) and phosphatidylethanolamine (PE) from natural headgroup extracts. When the two extracts are mixed, AmtB retains more species, wherein selectivity is tuned to bias headgroup selection. Using a series of natural headgroup extracts, we show TRAAK, a two-pore domain K+ channel (K2P), retains specific acyl chains that is independent of the headgroup. A brain polar lipid extract was then combined with the K2Ps, TRAAK and TREK2, to understand lipid specificity. More than a hundred lipids demonstrated affinity for each protein, and both channels were found to retain specific fatty acids and lysophospholipids known to stimulate channel activity, even after several column washes. Natural lipid extracts provide the unique opportunity to not only present natural lipid diversity to purified membrane proteins but also identify lipids that may be important for membrane protein structure and function.

Activation of Slack Potassium Channels (KCNT1) Triggers an Increase in mRNA Translation

The Slack ion channel is a member of a family of large conductance sodium-activated potassium channels. It is expressed predominantly in neurons of the central nervous system where it regulates neuronal excitability. FMRP, an important regulator of mRNA translation, binds both Slack mRNA and the Slack protein. The association of Slack with FMRP stimulates channel activity, raising the possibility that activation of Slack channels may also regulate the function of FMRP. Our laboratory has previously identified Slack as required for a translation-dependent recovery from an extended period of inhibition in Aplysia neurons following stimulation, further suggesting that Slack may play a role in the regulation of mRNA translation. Here we provide the initial evidence for such a role. We transfected cells with a fluorescent reporter for mRNA translation, which contains the 5’ and 3’ sequences of the mRNA for β-actin, but with the coding region replaced with that for the irreversibly-photoconvertible fluorescent protein dendra2. Using experiments that visualize real-time translation in a stable Slack-expressing HEK cell line and in mouse cortical neurons, together with pharmacological manipulation and silencing RNA knockdowns, we have found that Slack activation causes an increase in translation that is enhanced in the absence of FMRP. This increase in translation persists in the presence of the Slack channel blocker quinidine, indicating that it does not require ion flux through the channel. Puromycin incorporation experiments suggest Slack activation may produce a global increase in translation. Additional experiments in HEK cell culture suggest that Slack binding partners CYFIP1, another FMRP binding protein; and Phactr-1, a PP1 binding protein, may also modulate this channel-dependent translation. This mechanism of Slack-dependent translation potentially represents the first instance of the direct modulation of mRNA translation by activation of an ion channel.

TRPV4 and KRAS and FGFR1 gain-of-function mutations drive giant cell lesions of the jaw

Giant cell lesions of the jaw (GCLJ) are debilitating tumors of unknown origin with limited available therapies. Here, we analyze 58 sporadic samples using next generation or targeted sequencing and report somatic, heterozygous, gain-of-function mutations in KRAS, FGFR1, and p.M713V/I-TRPV4 in 72% (42/58) of GCLJ. TRPV4 p.M713V/I mutations are exclusive to central GCLJ and occur at a critical position adjacent to the cation permeable pore of the channel. Expression of TRPV4 mutants in HEK293 cells leads to increased cell death, as well as increased constitutive and stimulated channel activity, both of which can be prevented using TRPV4 antagonists. Furthermore, these mutations induce sustained activation of ERK1/2, indicating that their effects converge with that of KRAS and FGFR1 mutations on the activation of the MAPK pathway in GCLJ. Our data extend the spectrum of TRPV4 channelopathies and provide rationale for the use of TRPV4 and RAS/MAPK antagonists at the bedside in GCLJ.

Structural basis for PtdInsP2-mediated human TRPML1 regulation

Transient receptor potential mucolipin 1 (TRPML1), a lysosomal channel, maintains the low pH and calcium levels for lysosomal function. Several small molecules modulate TRPML1 activity. ML-SA1, a synthetic agonist, binds to the pore region and phosphatidylinositol-3,5-bisphosphate (PtdIns(3,5)P2), a natural lipid, stimulates channel activity to a lesser extent than ML-SA1; moreover, PtdIns(4,5)P2, another natural lipid, prevents TRPML1-mediated calcium release. Notably, PtdIns(3,5)P2 and ML-SA1 cooperate further increasing calcium efflux. Here we report the structures of human TRPML1 at pH 5.0 with PtdIns(3,5)P2, PtdIns(4,5)P2, or ML-SA1 and PtdIns(3,5)P2, revealing a unique lipid-binding site. PtdIns(3,5)P2 and PtdIns(4,5)P2 bind to the extended helices of S1, S2, and S3. The phosphate group of PtdIns(3,5)P2 induces Y355 to form a π-cation interaction with R403, moving the S4–S5 linker, thus allosterically activating the channel. Our structures and electrophysiological characterizations reveal an allosteric site and provide molecular insight into how lipids regulate TRP channels.

Upregulation of basolateral small conductance potassium channels (KCNQ1/KCNE3) in ulcerative colitis

BackgroundBasolateral K+ channels hyperpolarize colonocytes to ensure Na+ (and thus water) absorption. Small conductance basolateral (KCNQ1/KCNE3) K+ channels have never been evaluated in human colon. We therefore evaluated KCNQ1/KCNE3 channels in distal colonic crypts obtained from normal and active ulcerative colitis (UC) patients. MethodsKCNQ1 and KCNE3 mRNA levels were determined by qPCR, and KCNQ1/KCNE3 channel activity in normal and UC crypts, and the effects of forskolin (activator of adenylate cyclase) and UC-related proinflammatory cytokines on normal crypts, studied by patch clamp recording. ResultsWhereas KCNQ1 and KCNE3 mRNA expression was similar in normal and UC crypts, single 6.8 pS channels were seen in 36% of basolateral patches in normal crypts, and to an even greater extent (74% of patches, P < 0.001) in UC crypts, with two or more channels per patch. Channel activity was 10-fold higher (P < 0.001) in UC crypts, with a greater contribution to basolateral conductance (5.85 ± 0.62 mS cm−2) than in controls (0.28 ± 0.04 mS cm−2, P < 0.001). In control crypts, forskolin and thromboxane A2 stimulated channel activity 30-fold and 10-fold respectively, while PGE2, IL-1β, and LTD4 had no effect. ConclusionsKCNQ1/KCNE3 channels make only a small contribution to basolateral conductance in normal colonic crypts, with increased channel activity in UC appearing insufficient to prevent colonic cell depolarization in this disease. This supports the proposal that defective Na+ absorption rather than enhanced Cl− secretion, is the dominant pathophysiological mechanism of diarrhea in UC.

The Polarized Effect of Intracellular Calcium on the Renal Epithelial Sodium Channel Occurs as a Result of Subcellular Calcium Signaling Domains Maintained by Mitochondria

The renal epithelial sodium channel (ENaC) provides regulated sodium transport in the distal nephron. The effects of intracellular calcium ([Ca(2+)]i) on this channel are only beginning to be elucidated. It appears from previous studies that the [Ca(2+)]i increases downstream of ATP administration may have a polarized effect on ENaC, where apical application of ATP and the subsequent [Ca(2+)]i increase have an inhibitory effect on the channel, whereas basolateral ATP and [Ca(2+)]i have a stimulatory effect. We asked whether this polarized effect of ATP is, in fact, reflective of a polarized effect of increased [Ca(2+)]i on ENaC and what underlying mechanism is responsible. We began by performing patch clamp experiments in which ENaC activity was measured during apical or basolateral application of ionomycin to increase [Ca(2+)]i near the apical or basolateral membrane, respectively. We found that ENaC does indeed respond to increased [Ca(2+)]i in a polarized fashion, with apical increases being inhibitory and basolateral increases stimulating channel activity. In other epithelial cell types, mitochondria sequester [Ca(2+)]i, creating [Ca(2+)]i signaling microdomains within the cell that are dependent on mitochondrial localization. We found that mitochondria localize in bands just beneath the apical and basolateral membranes in two different cortical collecting duct principal cell lines and in cortical collecting duct principal cells in mouse kidney tissue. We found that inhibiting mitochondrial [Ca(2+)]i uptake destroyed the polarized response of ENaC to [Ca(2+)]i. Overall, our data suggest that ENaC is regulated by [Ca(2+)]i in a polarized fashion and that this polarization is maintained by mitochondrial [Ca(2+)]i sequestration.

Tu1390 Anoctamin 6 Contributes to Accessory Cholera Enterotoxin (ACE) Stimulated Chloride Secretion in Intestinal Epithelial Cells to Induced Diarrhea

) than in control patients (0.28 ± 0.04 mS.cm-2 , P<0.001), but this increase in GSK did not compensate for the decrease in basolateral membrane conductance (from 22 mS.cm-2 to 5 mS.cm -2 ) previously attributed to decreased IK channel expression/activity in UC (J Pathol 2012;226:463-470). In control crypts, 10μM forskolin and 1μM thromboxane A2 stimulated channel activity 30-fold (n=8, P<0.005) and 10-fold (n=5, P<0.05) respectively, while 100nM PGE 2, 10nM IL-1β and 1μM LTD4 had no effect. Conclusions: Crypt cells in normal human colon express cAMP-activated KCNQ1/ KCNE3 channels which make a far smaller contribution to overall basolateral conductance than IK channels. In active UC, KCNQ1/KCNE3 channel activity is significantly increased, but this fails to compensate for the depolarizing effect of decreased IK channel expression/ activity. This may explain, at least in part, why defective electrogenic Na+ absorption rather than increased electrogenic Cl˜- secretion is the dominant pathophysiological mechanism of diarrhea in active UC (J Pathol 2012;226:463-470).

Tu1389 Increased Basolateral Small Conductance Potassium Channel (KCNQ1/ KCNE3) Activity in Ulcerative Colitis

Introduction and Aims: Basolateral K+ channels keep colonic cells in a hyperpolarized state to ensure transcellular ionic movements. Intermediate conductance K+ (IK) channels dominate the basolateral membrane conductance in human colonic cells, but their expression and activity are markedly decreased in ulcerative colitis (UC), thus contributing to defective Na+ and water absorption across inflamed mucosa (J Pathol 2012;226:463-470). This study is the first to characterize basolateral small conductance (KCNQ1/KCNE3) K+ channels in normal human colon, and explore their activity in UC. Methods: Intact crypts were isolated from distal colonic biopsies taken while investigating patients with functional diarrhea (normals) and patients with UC not receiving steroids. Expression of KCNQ1 and KCNE3 mRNAs was determined by qPCR. Patch clamp recording was used to study (i) basolateral KCNQ1/KCNE3 channel activity in normal and UC crypt cells and (ii) in normal crypts cells, the effects of forskolin (an activator of adenylate cyclase) and inflammatory mediators present at high concentrations in UC. Results: qPCR product bands were of the expected size (KCNQ1 160 bp, KCNE3 119 bp, β-actin 148 bp). Product identity was confirmed by automated sequencing and melting curve analysis, and KCNQ1 and KCNE3 mRNA expression was similar in normal and UC patients. In normal crypts (n=34), usually single channels (conductance 6.8 ± 0.5 pS) were seen in 36% of patches, whereas in UC crypts (n=9), two or more channels were seen in 74% of patches, the difference in channel prevalence between the two groups being significant (P<0.001). Overall channel activity was 10-fold greater in UC than control patients (P<0.001). These channels constituted a greater calculated basolateral conductance (GSK) in UC (5.85 ± 0.62 mS.cm-2) than in control patients (0.28 ± 0.04 mS.cm-2, P<0.001), but this increase in GSK did not compensate for the decrease in basolateral membrane conductance (from 22 mS.cm-2 to 5 mS.cm-2) previously attributed to decreased IK channel expression/activity in UC (J Pathol 2012;226:463-470). In control crypts, 10μM forskolin and 1μM thromboxane A2 stimulated channel activity 30-fold (n=8, P<0.005) and 10-fold (n=5, P<0.05) respectively, while 100nM PGE2, 10nM IL-1β and 1μM LTD4 had no effect. Conclusions: Crypt cells in normal human colon express cAMP-activated KCNQ1/ KCNE3 channels which make a far smaller contribution to overall basolateral conductance than IK channels. In active UC, KCNQ1/KCNE3 channel activity is significantly increased, but this fails to compensate for the depolarizing effect of decreased IK channel expression/ activity. This may explain, at least in part, why defective electrogenic Na+ absorption rather than increased electrogenic Cl secretion is the dominant pathophysiological mechanism of diarrhea in active UC (J Pathol 2012;226:463-470).

Reinterpreting the Action of ATP Analogs on KATP Channels

Neuroendocrine-type K(ATP) channels, (SUR1/Kir6.2)4, couple the transmembrane flux of K(+), and thus membrane potential, with cellular metabolism in various cell types including insulin-secreting β-cells. Mutant channels with reduced activity are a cause of congenital hyperinsulinism, whereas hyperactive channels are a cause of neonatal diabetes. A current regulatory model proposes that ATP hydrolysis is required to switch SUR1 into post-hydrolytic conformations able to antagonize the inhibitory action of nucleotide binding at the Kir6.2 pore, thus coupling enzymatic and channel activities. Alterations in SUR1 ATPase activity are proposed to contribute to neonatal diabetes and type 2 diabetes risk. The regulatory model is partly based on the reduced ability of ATP analogs such as adenosine 5'-(β,γ-imino)triphosphate (AMP-PNP) and adenosine 5'-O-(thiotriphosphate) (ATPγS) to stimulate channel activity, presumably by reducing hydrolysis. This study uses a substitution at the catalytic glutamate, SUR(1E1507Q), with a significantly increased affinity for ATP, to probe the action of these ATP analogs on conformational switching. ATPγS, a slowly hydrolyzable analog, switches SUR1 conformations, albeit with reduced affinity. Nonhydrolyzable AMP-PNP and adenosine 5'-(β,γ-methylenetriphosphate) (AMP-PCP) alone fail to switch SUR1, but do reverse ATP-induced switching. AMP-PCP displaces 8-azido-[(32)P]ATP from the noncanonical NBD1 of SUR1. This is consistent with structural data on an asymmetric bacterial ABC protein that shows that AMP-PNP binds selectively to the noncanonical NBD to prevent conformational switching. The results imply that MgAMP-PNP and MgAMP-PCP (AMP-PxP) fail to activate K(ATP) channels because they do not support NBD dimerization and conformational switching, rather than by limiting enzymatic activity.

Regulation of K Channels by the G Protein Signaling System

Over 25 years ago G protein inwardly rectifying K+ (GIRK) channels were the first effector shown to be activated by the βγ subunits of G proteins (Gβγ). Perfusion of atrial inside-out patches with purified Gβγ subunits stimulated channel activity even when the cells had been treated with pertussis toxin that functionally uncouples endogenous G proteins from receptor signaling. Furthermore, the lack of stimulation of channel activity by the Gα subunits led to the proposal that Gβγ independently activates GIRK channels (Logothetis et al., 1987). A decade later it was appreciated that GIRK channels required the presence of phosphoinositides, such as PIP2, in order to be gated by Gβγ subunits or other gating molecules such as intracellular Na+ or alcohols (Sui et al., 1998). In the following decade through the present times, crystal structures of the GIRK channels have been elucidated revealing the architecture of full-length GIRK channels in the absence or presence of PIP2 (Nishida et al., 1997; Whorton et al., 2011). Although attempts to obtain structures of the channels in complex with Gβγ have not succeeded thus far, insights on the sites of Gβγ and PIP2 action leading to channel gating are beginning to emerge. Seeking to provide a more rigorous test of the hypothesis that Gβγ stimulates GIRK channel activity independently of the remaining components of the system, we have successfully reconstituted the entire G protein signaling complex with the channel into planar lipid bilayers. To our surprise the signaling complex functioned equivalently in the bilayer as it did in excised patches only when the entire complex was reconstituted (i.e. receptor, heterotrimeric G proteins, and GIRK channel). The results obtained in the bilayer system put in question the hypothesis that Gβγ stimulates channel activity independently of the other components of the signaling complex.

Renal cyst fluid from human polycystic kidney disease patients stimulates Cl − transport

Autosomal dominant polycystic kidney disease (ADPKD) is characterized by the slow growth of fluid-filled cysts predominately in the kidney and in liver bile ducts. The factors involved in modifying the rate of cyst growth through epithelial proliferation or secretion are critical to understanding the progression of the disease. In addition, elucidation of mechanisms that potentiate the normal progression to renal failure will provide the basis for therapeutic intervention. Of note are the observations that the decline in renal function in middle age is precipitous and that renal injury results in an exacerbation of cyst growth. Using electrophysiological and biochemical techniques, we identified LPA (lysophosphatic acid) as a component of cyst fluid that stimulates secretory Cl− transport via two anion channels, CFTR and TMEM16a, in the mpkCCDcl4 model of renal principal cells. The LPA effect is manifested through receptors located on the basolateral membrane of polarized renal cells resulting in stimulation of channel activity in the apical membrane. Concentrations of LPA measured in ADPKD cyst fluid and in normal serum are sufficient to maximally stimulate ion transport. Thus, cyst fluid seepage into the interstitial space and/or leakage of vascular LPA are capable of stimulating epithelial cell secretion resulting in cyst enlargement. Research Support: IUPUI Membrane Biosciences Signature Center Grant

Molecular Mechanism of Calcium Channel Regulation in the Fight-Or-Flight Response

During the fight-or-flight response, the sympathetic nervous system stimulates L-type calcium currents in the heart conducted by Cav1.2 channels through activation of β-adrenergic receptors, adenylyl cyclase, and phosphorylation by cAMP-dependent protein kinase (PKA), thereby increasing cardiac contractility and beat rate. The channel α1 subunit C-terminus contains binding sites for multiple regulatory proteins including the PKA/A kinase anchoring protein 15 (AKAP15) complex. The C-terminus is proteolytically cleaved but remains associated non-covalently with the truncated channel and acts as a potent autoinhibitor of channel activity. Relief of this autoinhibition provides an attractive mechanism for cellular regulatory signals to produce the large increases in calcium current observed physiologically. We reconstituted regulation of Cav1.2 channels in non-muscle tsA-201 cells by forming an autoregulatory signaling complex composed of the Cav1.2Δ1800 channel, the noncovalently-associated distal C-terminal domain, the auxiliary α2δ1 and β2b subunits, and AKAP15. During whole-cell recordings of channel activity we observed a 3.6-fold range of Cav1.2 activity from a minimum in the presence of protein kinase inhibitors to a maximum with activation of adenylyl cyclase with forskolin. Equivalent modulation was not observed for the full-length (untruncated) channel or the truncated channel without the distal C-terminus. Basal channel activity in unstimulated cells was regulated by phosphorylation of two novel sites at Ser1700 in a PKA consensus sequence and Thr1704 in a casein kinase 2 consensus sequence, both strategically located at the interface between the distal and proximal C-terminal regulatory domains. Further stimulation of channel activity via PKA signaling required only phosphorylation of Ser1700. Phosphorylation at Ser1928 did not significantly alter channel activity. These results define the signaling complex required for Cav1.2 channel regulation and elucidate the sites of phosphorylation that regulate channel activity.Supported by NIH grants R01 HL085372, T32 HL007312-31 and AHA fellowship 09POST2080270

Molecular mechanism of calcium channel regulation in the fight-or-flight response.

During the fight-or-flight response, the sympathetic nervous system stimulates L-type calcium ion (Ca2+) currents conducted by Ca(V)1 channels through activation of β-adrenergic receptors, adenylyl cyclase, and phosphorylation by adenosine 3',5'-monophosphate-dependent protein kinase [also known as protein kinase A (PKA)], increasing contractility of skeletal and cardiac muscles. We reconstituted this regulation of cardiac Ca(V)1.2 channels in non-muscle cells by forming an autoinhibitory signaling complex composed of Ca(V)1.2Δ1800 (a form of the channel truncated at the in vivo site of proteolytic processing), its noncovalently associated distal carboxyl-terminal domain, the auxiliary α₂δ₁ and β(2b) subunits, and A-kinase anchoring protein 15 (AKAP15). A factor of 3.6 range of Ca(V)1.2 channel activity was observed from a minimum in the presence of protein kinase inhibitors to a maximum upon activation of adenylyl cyclase. Basal Ca(V)1.2 channel activity in unstimulated cells was regulated by phosphorylation of serine-1700 and threonine-1704, two residues located at the interface between the distal and the proximal carboxyl-terminal regulatory domains, whereas further stimulation of channel activity through the PKA signaling pathway only required phosphorylation of serine-1700. Our results define a conceptual framework for Ca(V)1.2 channel regulation and identify sites of phosphorylation that regulate channel activity.

Identification of a Thiol/Disulfide Redox Switch in the Human BK Channel That Controls Its Affinity for Heme and CO

Heme is a required prosthetic group in many electron transfer proteins and redox enzymes. The human BK channel, which is a large-conductance Ca(2+) and voltage-activated K(+) channel, is involved in the hypoxic response in the carotid body. The BK channel has been shown to bind and undergo inhibition by heme and activation by CO. Furthermore, evidence suggests that human heme oxygenase-2 (HO2) acts as an oxygen sensor and CO donor that can form a protein complex with the BK channel. Here we describe a thiol/disulfide redox switch in the human BK channel and biochemical experiments of heme, CO, and HO2 binding to a 134-residue region within the cytoplasmic domain of the channel. This region, called the heme binding domain (HBD) forms a linker segment between two Ca(2+)-sensing domains (called RCK1 and RCK2) of the BK channel. The HBD includes a CXXCH motif in which histidine serves as the axial heme ligand and the two cysteine residues can form a reversible thiol/disulfide redox switch that regulates affinity of the HBD for heme. The reduced dithiol state binds heme (K(d) = 210 nm) 14-fold more tightly than the oxidized disulfide state. Furthermore, the HBD is shown to tightly bind CO (K(d) = 50 nm) with the Cys residues in the CXXCH motif regulating affinity of the HBD for CO. This HBD is also shown to interact with heme oxygenase-2. We propose that the thiol/disulfide switch in the HBD is a mechanism by which activity of the BK channel can respond quickly and reversibly to changes in the redox state of the cell, especially as it switches between hypoxic and normoxic conditions.

Interaction between mutations in the slide helix of Kir6.2 associated with neonatal diabetes and neurological symptoms

ATP-sensitive potassium (K(ATP)) channels regulate insulin secretion from pancreatic beta-cells. Gain-of-function mutations in the genes encoding the Kir6.2 and SUR1 subunits of this channel cause neonatal diabetes. We report two novel mutations on the same haplotype (cis), F60Y and V64L, in the slide helix of Kir6.2 in a patient with neonatal diabetes, developmental delay and epilepsy. Functional analysis revealed the F60Y mutation increases the intrinsic channel open probability (Po(0)), thereby indirectly producing a marked decrease in channel inhibition by ATP and an increase in whole-cell K(ATP) currents. When expressed alone, the V64L mutation caused a small reduction in apparent ATP inhibition, by enhancing the ability of MgATP to stimulate channel activity. The V64L mutation also ameliorated the deleterious effects on the F60Y mutation when it was expressed on the same (but not a different) subunit. These data indicate that F60Y is the pathogenic mutation and reveal that interactions between slide helix residues can influence K(ATP) channel gating.

Molecular biology of KATP channels and implications for health and disease

The ATP-sensitive potassium (K(ATP)) channel is expressed in most excitable tissues and plays a critical role in numerous physiological processes by coupling intracellular energetics to electrical activity. The channel is comprised of four Kir6.x subunits associated with four regulatory sulfonylurea receptors (SUR). Intracellular ATP acts on Kir6.x to inhibit channel activity, while MgADP stimulates channel activity through SUR. Changes in the cytosolic [ATP] to [ADP] ratio thus determine channel activity. Multiple mutations in Kir6.x and SUR genes have implicated K(ATP) channels in various diseases ranging from diabetes and hyperinsulinism to cardiac arrhythmias and cardiovascular disease. Continuing studies of channel physiology and pathology will bring new insights to the molecular basis of K(ATP) channel function, leading to a better understanding of the role that K(ATP) channels play in both health and disease.

Heme Oxygenase Induced Endothelial BKCa Channel Activity Following Chronic Hypoxia

Exposure to chronic hypoxia (CH) results in blunted myogenic and agonist‐induced tone in systemic arteries that persists upon return to normoxia. This impaired vasoconstrictor reactivity is reversed by either removal of the endothelium or by blockade of the CO producing enzyme heme oxygenase (HO). Interestingly, endothelial cell (EC) specific blockade of large conductance Ca2+‐activated K+ (BKCa) channels also restores vasoreactivity in arteries from CH rats. Patch clamp studies demonstrate EC BKCa activity following CH, but not in ECs from control rats, although channels are observed by immunofluorescence (IF) in cells from both groups. Since HO‐derived CO is an endogenous activator of BKCa channels, we hypothesized that HO and BKCa form a functional unit that results in enhanced channel activity following CH. In support of the hypothesis, the HO inhibitor zinc protoporphyrin IX (ZnPPIX) significantly decreased K+ currents in ECs from CH rats, but was without effect in EC from control rats. In addition, the HO substrate hemin increased K+ currents only in the CH group. HO‐2, but not HO‐1, was found to co‐localize with BKCa in cells from each group. These results suggest that although HO and BKCa channels are expressed in EC from each group, HO‐derived CO only stimulates channel activity following CH.

Role of individual R domain phosphorylation sites in CFTR regulation by protein kinase A

The cystic fibrosis transmembrane conductance regulator (CFTR) plays a critical role in transcellular ion transport and when defective, results in the genetic disease cystic fibrosis. CFTR is novel in the ATP-binding cassette superfamily as an ion channel that is enabled by a unique unstructured regulatory domain. This R domain contains multiple protein kinase A sites, which when phosphorylated allow channel gating. Most of the sites have been indicated to stimulate channel activity, while two of them have been suggested to be inhibitory. It is unknown whether individual sites act coordinately or distinctly. To address this issue, we raised monoclonal antibodies recognizing the unphosphorylated, but not the phosphorylated states of four functionally relevant sites (700, 737, 768, and 813). This enabled simultaneous monitoring of their phosphorylation and dephosphorylation and revealed that both processes occurred rapidly at the first three sites, but more slowly at the fourth. The parallel phosphorylation rates of the stimulatory 700 and the putative inhibitory 737 and 768 sites prompted us to reexamine the role of the latter two. With serines 737 and 768 reintroduced individually into a PKA insensitive variant, in which serines at 15 sites had been replaced by alanines, a level of channel activation by PKA was restored, showing that these sites can mediate stimulation. Thus, we have provided new tools to study the CFTR regulation by phosphorylation and found that sites proposed to inhibit channel activity can also participate in stimulation.