Qadeer Aziz

The role of ATP‐sensitive potassium channels in cellular function and protection in the cardiovascular system

ATP‐sensitive potassium channels (KATP) are widely distributed and present in a number of tissues including muscle, pancreatic beta cells and the brain. Their activity is regulated by adenine nucleotides, characteristically being activated by falling ATP and rising ADP levels. Thus, they link cellular metabolism with membrane excitability. Recent studies using genetically modified mice and genomic studies in patients have implicated KATP channels in a number of physiological and pathological processes. In this review, we focus on their role in cellular function and protection particularly in the cardiovascular system.

Molecular mechanism of voltage sensor movements in a potassium channel

Voltage‐gated potassium channels are six‐transmembrane (S1–S6) proteins that form a central pore domain (4 × S5–S6) surrounded by four voltage sensor domains (S1–S4), which detect changes in membrane voltage and control pore opening. Upon depolarization, the S4 segments move outward carrying charged residues across the membrane field, thereby leading to the opening of the pore. The mechanism of S4 motion is controversial. We have investigated how S4 moves relative to the pore domain in the prototypical Shaker potassium channel. We introduced pairs of cysteines, one in S4 and the other in S5, and examined proximity changes between each pair of cysteines during activation, using Cd2+ and copper‐phenanthroline, which crosslink the cysteines with metal and disulphide bridges, respectively. Modelling of the results suggests a novel mechanism: in the resting state, the top of the S3b–S4 voltage sensor paddle lies close to the top of S5 of the adjacent subunit, but moves towards the top of S5 of its own subunit during depolarization—this motion is accompanied by a reorientation of S4 charges to the extracellular phase.

The ATP-Sensitive Potassium Channel Subunit, Kir6.1, in Vascular Smooth Muscle Plays a Major Role in Blood Pressure Control

ATP-sensitive potassium channels (KATP) regulate a range of biological activities by coupling membrane excitability to the cellular metabolic state. In particular, it has been proposed that KATP channels and specifically, the channel subunits Kir6.1 and SUR2B, play an important role in the regulation of vascular tone. However, recent experiments have suggested that KATP channels outside the vascular smooth muscle compartment are the key determinant of the observed behavior. Thus, we address the importance of the vascular smooth muscle KATP channel, using a novel murine model in which it is possible to conditionally delete the Kir6.1 subunit. Using a combination of molecular, electrophysiological, in vitro, and in vivo techniques, we confirmed the absence of Kir6.1 and KATP currents and responses specifically in smooth muscle. Mice with conditional deletion of Kir6.1 showed no obvious arrhythmic phenotype even after provocation with ergonovine. However, these mice were hypertensive and vascular smooth muscle cells failed to respond to vasodilators in a normal fashion. Thus, Kir6.1 underlies the vascular smooth muscle KATP channel and has a key role in vascular reactivity and blood pressure control.

Regulation of the ATP-sensitive potassium channel subunit, Kir6.2, by a Ca2+-dependent protein kinase C.

The activity of ATP-sensitive potassium (KATP) channels is governed by the concentration of intracellular ATP and ADP and is thus responsive to the metabolic status of the cell. Phosphorylation of KATP channels by protein kinase A (PKA) or protein kinase C (PKC) results in the modulation of channel activity and is particularly important in regulating smooth muscle tone. At the molecular level the smooth muscle channel is composed of a sulfonylurea subunit (SUR2B) and a pore-forming subunit Kir6.1 and/or Kir6.2. Previously, Kir6.1/SUR2B channels have been shown to be inhibited by PKC, and Kir6.2/SUR2B channels have been shown to be activated or have no response to PKC. In this study we have examined the modulation of channel complexes formed of the inward rectifier subunit, Kir6.2, and the sulfonylurea subunit, SUR2B. Using a combination of biochemical and electrophysiological techniques we show that this complex can be inhibited by protein kinase C in a Ca2+-dependent manner and that this inhibition is likely to be as a result of internalization. We identify a residue in the distal C terminus of Kir6.2 (Ser-372) whose phosphorylation leads to down-regulation of the channel complex. This inhibitory effect is distinct from activation which is seen with low levels of channel activity.

Potential role of reduced basolateral potassium (IKCa3.1) channel expression in the pathogenesis of diarrhoea in ulcerative colitis.

Diarrhoea in ulcerative colitis (UC) mainly reflects impaired colonic Na+ and water absorption. Colonocyte membrane potential, an important determinant of electrogenic Na+ absorption, is reduced in UC. Colonocyte potential is principally determined by basolateral IK (KCa3.1) channel activity. To determine whether reduced Na+ absorption in UC might be associated with decreased IK channel expression and activity, we used molecular and patch clamp recording techniques to evaluate IK channels in colon from control patients and patients with active UC. In control patients, immunolabelling revealed basolateral IK channels distributed uniformly along the surface‐crypt axis, with substantially decreased immunolabelling in patients with active UC, although IK mRNA levels measured by quantitative PCR were similar in both groups. Patch clamp analysis indicated that cell conductance was dominated by basolateral IK channels in control patients, but channel abundance and overall activity were reduced by 53% (p = 0.03) and 61% (p = 0.04), respectively, in patients with active UC. These changes resulted in a 75% (p = 0.003) decrease in the estimated basolateral membrane K+ conductance in UC patients compared with controls. Levels of IK channel immunolabelling and activity in UC patients in clinical remission were similar to those in control patients. We conclude that a substantial decrease in basolateral IK channel expression and activity in active UC most likely explains the epithelial cell depolarization observed in this disease, and decreases the electrical driving force for electrogenic Na+ transport, thereby impairing Na+ absorption (and as a consequence, Cl− and water absorption) across the inflamed mucosa. Copyright

Molecular and functional characterization of the endothelial ATP-sensitive potassium channel

ATP-sensitive potassium (KATP) channels are widely expressed in the cardiovascular system, where they regulate a range of biological activities by linking cellular metabolism with membrane excitability. KATP channels in vascular smooth muscle have a well-defined role in regulating vascular tone. KATP channels are also thought to be expressed in vascular endothelial cells, but their presence and function in this context are less clear. As a result, we aimed to investigate the molecular composition and physiological role of endothelial KATP channels. We first generated mice with an endothelial specific deletion of the channel subunit Kir6.1 (eKO) using cre-loxP technology. Data from qRT-PCR, patch clamp, ex vivo coronary perfusion Langendorff heart experiments, and endothelial cell Ca2+ imaging comparing eKO and wild-type mice show that Kir6.1-containing KATP channels are indeed present in vascular endothelium. An increase in intracellular [Ca2+], which is central to changes in endothelial function such as mediator release, at least partly contributes to the endothelium-dependent vasorelaxation induced by the KATP channel opener pinacidil. The absence of Kir6.1 did not elevate basal coronary perfusion pressure in eKO mice. However, vasorelaxation was impaired during hypoxia in the coronary circulation, and this resulted in greater cardiac injury during ischemia–reperfusion. The response to adenosine receptor stimulation was impaired in eKO mice in single cells in patch clamp recordings and in the intact coronary circulation. Our data support the existence of an endothelial KATP channel that contains Kir6.1, is involved in vascular reactivity in the coronary circulation, and has a protective role in ischemia reperfusion.

Molecular mechanisms of congenital hyperinsulinism due to autosomal dominant mutations in ABCC8

Congenital Hyperinsulinism (CHI) is a rare heterogeneous disease characterized by unregulated insulin secretion. Dominant mutations in ABCC8 causing medically unresponsive CHI have been reported; however, the molecular mechanisms are not clear. The molecular basis of medically unresponsive CHI due to dominant ABCC8 mutations has been studied in 10 patients, who were medically unresponsive to diazoxide (DZX), and nine of whom required a near-total pancreatectomy, and one partial pancreatectomy. DNA sequencing revealed seven dominant inactivating heterozygous missense mutations in ABCC8, including one novel and six previously reported but uncharacterized mutations. Two groups of mutations with different cellular mechanisms were characterized. Mutations in the transmembrane domain (TMD) were more responsive to channel activators such as DZX, MgADP and metabolic inhibition. The trafficking analysis has shown that nucleotide-binding domain two (NBD2) mutations are not retained in the endoplasmic reticulum (ER) and are present on the membrane. However, the TMD mutations were retained in the ER. D1506E was the most severe SUR1-NBD2 mutation. Homologous expression of D1506E revealed a near absence of KATP currents in the presence of DZX and intracellular MgADP. Heterozygous expression of D1506E showed a strong dominant-negative effect on SUR1\Kir6.2 currents. Overall, we define two groups of mutation with different cellular mechanisms. In the first group, channel complexes with mutations in NBD2 of SUR1 traffic normally but are unable to be activated by MgADP. In the second group, channels mutations in the TMD of SUR1 are retained in the ER and have variable functional impairment.

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

Background Basolateral 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. Methods KCNQ1 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. Results Whereas 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. Conclusions KCNQ1/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.

ATP-sensitive potassium channels and vascular function.

ATP-sensitive potassium channels (KATP) are widely distributed in different organs of the body. They are best described in pancreatic β cells and cardiac myocytes but are also present in the periph...

ATP-sensitive potassium channels in the sinoatrial node contribute to heart rate control and adaptation to hypoxia

ATP-sensitive potassium channels (KATP) contribute to membrane currents in many tissues, are responsive to intracellular metabolism, and open as ATP falls and ADP rises. KATP channels are widely distributed in tissues and are prominently expressed in the heart. They have generally been observed in ventricular tissue, but they are also expressed in the atria and conduction tissues. In this study, we focused on the contribution and role of the inwardly rectifying KATP channel subunit, Kir6.1, in the sinoatrial node (SAN). To develop a murine, conduction-specific Kir6.1 KO model, we selectively deleted Kir6.1 in the conduction system in adult mice (cKO). Electrophysiological data in single SAN cells indicated that Kir6.1 underlies a KATP current in a significant proportion of cells and influences early repolarization during pacemaking, resulting in prolonged cycle length. Implanted telemetry probes to measure heart rate and electrocardiographic characteristics revealed that the cKO mice have a slow heart rate, with episodes of sinus arrest in some mice. The PR interval (time between the onset of the P wave to the beginning of QRS complex) was increased, suggesting effects on the atrioventricular node. Ex vivo studies of whole heart or dissected heart regions disclosed impaired adaptive responses of the SAN to hypoxia, and this may have had long-term pathological consequences in the cKO mice. In conclusion, Kir6.1-containing KATP channels in the SAN have a role in excitability, heart rate control, and the electrophysiological adaptation of the SAN to hypoxia.