Richard D. Rainbow

Distribution of Kir6.0 and SUR2 ATP-sensitive potassium channel subunits in isolated ventricular myocytes

The subcellular distribution of ATP-sensitive potassium (K(ATP)) channel subunits in rat-isolated ventricular myocytes was investigated using a panel of subunit-specific antisera. Kir6.1 subunits were associated predominantly with myofibril structures and were co-localized with the mitochondrial marker MitoFluor red (correlation coefficient (cc) = 0.63 +/- 0.05). Anti-Kir6.1 antibodies specifically recognized a polypeptide of 48 kDa in mitochondrial membrane fractions consistent with the presence of Kir6.1 subunits in this organelle. Both Kir6.2 and SUR2A subunits were distributed universally over the sarcolemma. Lower-intensity antibody-associated immunofluorescence was detected intracellularly, which was correlated with the distribution of MitoFluor red in both cases (cc, Kir6.2, 0.56 +/- 0.05; SUR2A, 0.61 +/- 0.06). A polypeptide of 40 kDa was recognized by anti-Kir6.2-subunit antibodies in western blots of both microsomal and mitochondrial membrane fractions consistent with the presence of this subunit in the sarcolemma and mitochondria. Similarly, SUR2A and SUR2B subunits were detected in western blots of microsomal membrane proteins consistent with a sarcolemmal localization for these polypeptides. SUR2B subunits were shown in confocal microscopy to co-localize strongly with t-tubules (cc, 0.81 +/- 0.05). Together, the results indicate that Kir6.2 and SUR2A subunits predominate in the sarcolemma of ventricular myocytes consistent with a Kir6.2/SUR2A-subunit combination in the sarcolemmal K(ATP)channel. Kir6.1, Kir6.2 and SUR2A subunits were demonstrated in mitochondria. Combinations of these subunits would not explain the reported pharmacology of the mitochondrial K(ATP) channel (Mol Pharmacol 59 (2001) 225) suggesting the possibility of further unidentified components of this channel.

Glucose reduces endothelin inhibition of voltage-gated potassium channels in rat arterial smooth muscle cells

Prolonged hyperglycaemia impairs vascular reactivity and inhibits voltage‐activated K+ (Kv) channels. We examined acute effects of altering glucose concentration on the activity and inhibition by endothelin‐1 (ET‐1) of Kv currents of freshly isolated rat arterial myocytes. Peak Kv currents recorded in glucose‐free solution were reversibly reduced within 200 s by increasing extracellular glucose to 4 mm. This inhibitory effect of glucose was abolished by protein kinase C inhibitor peptide (PKC‐IP), and Kv currents were further reduced in 10 mm glucose. In current‐clamped cells, membrane potentials were more negative in 4 than in 10 mm glucose. In 4 mm d‐glucose, 10 nm ET‐1 decreased peak Kv current amplitude at +60 mV from 23.5 ± 3.3 to 12.1 ± 3.1 pA pF−1 (n= 6, P < 0.001) and increased the rate of inactivation, decreasing the time constant around fourfold. Inhibition by ET‐1 was prevented by PKC‐IP. When d‐glucose was increased to 10 mm, ET‐1 no longer inhibited Kv current (n= 6). Glucose metabolism was required for prevention of ET‐1 inhibition of Kv currents, since fructose mimicked the effects of d‐glucose, while l‐glucose, sucrose or mannitol were without effect. Endothelin receptors were still functional in 10 mm d‐glucose, since pinacidil‐activated ATP‐dependent K+ (KATP) currents were reduced by 10 nm ET‐1. This inhibition was nearly abolished by PKC‐IP, indicating that endothelin receptors could still activate PKC in 10 mm d‐glucose. These results indicate that changes in extracellular glucose concentration within the physiological range can reduce Kv current amplitude and can have major effects on Kv channel modulation by vasoconstrictors.

Endothelin-I and angiotensin II inhibit arterial voltage-gated K+ channels through different protein kinase C isoenzymes.

AIMS Voltage-gated K+ (Kv) channels of arterial smooth muscle (ASM) modulate arterial tone and are inhibited by vasoconstrictors through protein kinase C (PKC). We aimed to determine whether endothelin-1 (ET-1) and angiotensin II (AngII), which cause similar inhibition of Kv, use the same signalling pathway and PKC isoenzyme to exert their effects on Kv and to compare the involvement of PKC isoenzymes in contractile responses to these agents. METHODS AND RESULTS Kv currents recorded using the patch clamp technique with freshly isolated rat mesenteric ASM cells were inhibited by ET-1 or AngII. Inclusion of a PKCepsilon inhibitor peptide in the intracellular solution substantially reduced inhibition by AngII, but did not affect that by ET-1. Kv inhibition by ET-1 was reduced by the conventional PKC inhibitor Gö 6976 but not by the PKCbeta inhibitor LY333531. Selective peptide inhibitors of PKCalpha and PKCepsilon were linked to a Tat carrier peptide to make them membrane permeable and used to show that inhibition of PKCalpha prevented ET-1 inhibition of Kv current, but did not affect that by AngII. In contrast, inhibition of PKCepsilon prevented Kv inhibition by AngII but not by ET-1. The Tat-linked inhibitor peptides were also used to investigate the involvement of PKCalpha and PKCepsilon in the contractile responses of mesenteric arterial rings, showing that ET-1 contractions were substantially reduced by inhibition of PKCalpha, but unaffected by inhibition of PKCepsilon. AngII contractions were unaffected by inhibition of PKCalpha but substantially reduced by inhibition of PKCepsilon. CONCLUSION ET-1 inhibits Kv channels of mesenteric ASM through activation of PKCalpha, while AngII does so through PKCepsilon. This implies that ET-1 and AngII target Kv channels of ASM through different pathways of PKC-interacting proteins, so each vasoconstrictor enables its distinct PKC isoenzyme to interact functionally with the Kv channel.

Intracellular Zinc Modulates Cardiac Ryanodine Receptor-mediated Calcium Release *

Background: In heart failure, the release of calcium becomes erratic leading to the generation of arrhythmias. Dysregulated Zn2+ homeostasis occurs in chronic heart failure. Results: Zn2+ can directly activate RyR2, removing the dependence of Ca2+ for channel activation. Conclusion: Zn2+ shapes Ca2+ dynamics by directly interacting with and modulating RyR2 function. Significance: This highlights a new role for Zn2+ in cardiac excitation-contraction coupling. Aberrant Zn2+ homeostasis is a hallmark of certain cardiomyopathies associated with altered contractile force. In this study, we addressed whether Zn2+ modulates cardiac ryanodine receptor gating and Ca2+ dynamics in isolated cardiomyocytes. We reveal that Zn2+ is a high affinity regulator of RyR2 displaying three modes of operation. Picomolar free Zn2+ concentrations potentiate RyR2 responses, but channel activation is still dependent on the presence of cytosolic Ca2+. At concentrations of free Zn2+ >1 nm, Zn2+ is the main activating ligand, and the dependence on Ca2+ is removed. Zn2+ is therefore a higher affinity activator of RyR2 than Ca2+. Millimolar levels of free Zn2+ were found to inhibit channel openings. In cardiomyocytes, consistent with our single channel results, we show that Zn2+ modulates both the frequency and amplitude of Ca2+ waves in a concentration-dependent manner and that physiological levels of Zn2+ elicit Ca2+ release in the absence of activating levels of cytosolic Ca2+. This highlights a new role for intracellular Zn2+ in shaping Ca2+ dynamics in cardiomyocytes through modulation of RyR2 gating.

SUR2A C‐terminal fragments reduce KATP currents and ischaemic tolerance of rat cardiac myocytes

C‐terminal fragments of the sulphonylurea receptor SUR2A can alter the functional expression of cloned ATP‐sensitive K+ channels (KATP). To investigate the protective role of KATP channels during metabolic stress we transfected SUR2A fragments into adult rat cardiac myocytes. A fragment comprising residues 1294–1358, the A‐fragment, reduced sarcolemmal KATP currents by over 85% after 2 days (pinacidil‐activated current densities were: vector alone 7.04 ± 1.22; and A‐fragment 0.94 ± 0.07 pA pF−1, n= 6,6, P < 0.001). An inactive fragment (1358–1545, current density 6.30 ± 0.85 pA pF−1, n= 6) was used as a control. During metabolic inhibition (CN and iodoacetate) of isolated myocytes stimulated at 1 Hz, the A‐fragment delayed action potential shortening and contractile failure, but accelerated rigor contraction and increased Ca2+ loading. On reperfusion, A‐fragment‐transfected cells also showed increased intracellular Ca2+ and the proportion of cells recovering contractile function was reduced from 40.0 to 9.5% (P < 0.01). The protective effect of pretreatment with 2,4‐dinitrophenol, measured from increased functional recovery and reduced Ca2+ loading, was abolished by the A‐fragment. Our data are consistent with a role for KATP channels in causing action potential failure and reduced Ca2+ loading during metabolic stress, and with a major role in protection by preconditioning. The effects of the A‐fragment may arise entirely from reduced expression of the sarcolemmal KATP channel, but we also discuss the possibility of mitochondrial effects.

The sarcoplasmic reticulum Ca2+ store arrangement in vascular smooth muscle

n vascular smooth muscle cells, Ca2+ release via IP(3) receptors (IP(3)R) and ryanodine receptors (RyR) on the sarcoplasmic reticulum (SR) Ca2+ store contributes significantly to the regulation of cellular events such as gene regulation, growth and contraction. Ca2+ release from various regions of a structurally compartmentalized SR, it is proposed, may selectively activate different cellular functions. Multiple SR compartments with various receptor arrangements are proposed also to exist at different stages of smooth muscle development and in proliferative vascular diseases such as atherosclerosis. The conclusions on SR organization have been derived largely from the outcome of functional studies. This study addresses whether the SR Ca2+ store is a single continuous interconnected network or multiple separate Ca2+ pools in single vascular myocytes. To do this, the consequences of depletion of the SR in small restricted regions on the Ca2+ available throughout the store was examined using localized photolysis of caged-IP3 and focal application of ryanodine in guinea-pig voltage-clamped single portal vein myocytes. From one small site on the cell, the entire SR could be depleted via either RyR or IP(3)R. The entire SR could also be refilled from one small site on the cell. The results suggest a single luminally continuous SR exists. However, the opening of IP(3)R and RyR was regulated by the Ca2+ concentration within the SR (luminal [Ca2+]). As the luminal [Ca2+] declines, the opening of the receptors decline and stop, and there may appear to be stores with either only RyR or only IP(3)R. The SR Ca2+ store is a single luminally continuous entity which contains both IP(3)R and RyR and within which Ca2+ is accessed freely by each receptor. While the SR is a single continuous entity, regulation of IP3R and RyR by luminal [Ca2+] explains the appearance of multiple stores in some functional studies.

Proximal C-terminal domain of sulphonylurea receptor 2A interacts with pore-forming Kir6 subunits in KATP channels

Functional KATP (ATP-sensitive potassium) channels are hetero-octamers of four Kir6 (inwardly rectifying potassium) channel subunits and four SUR (sulphonylurea receptor) subunits. Possible interactions between the C-terminal domain of SUR2A and Kir6.2 were investigated by co-immunoprecipitation of rat SUR2A C-terminal fragments with full-length Kir6.2 and by analysis of cloned KATP channel function and distribution in HEK-293 cells (human embryonic kidney 293 cells) in the presence of competing rSUR2A fragments. Three maltose-binding protein-SUR2A fusions, rSUR2A-CTA (rSUR2A residues 1254-1545), rSUR2A-CTB (residues 1254-1403) and rSUR2A-CTC (residues 1294-1403), were co-immunoprecipitated with full-length Kir6.2 using a polyclonal anti-Kir6.2 antiserum. A fourth C-terminal domain fragment, rSUR2A-CTD (residues 1358-1545) did not co-immunoprecipitate with Kir6.2 under the same conditions, indicating a direct interaction between Kir6.2 and a 65-amino-acid section of the cytoplasmic C-terminal region of rSUR2A between residues 1294 and 1358. ATP- and glibenclamide-sensitive K+ currents were decreased in HEK-293 cells expressing full-length Kir6 and SUR2 subunits that were transiently transfected with fragments rSUR2A-CTA, rSUR2A-CTC and rSUR2A-CTE (residues 1294-1359) compared with fragment rSUR2A-CTD or mock-transfected cells, suggesting either channel inhibition or a reduction in the number of functional KATP channels at the cell surface. Anti-KATP channel subunit-associated fluorescence in the cell membrane was substantially lower and intracellular fluorescence increased in rSUR2A-CTE expressing cells; thus, SUR2A fragments containing residues 1294-1358 reduce current by decreasing the number of channel subunits in the cell membrane. These results identify a site in the C-terminal domain of rSUR2A, between residues 1294 and 1358, whose direct interaction with full-length Kir6.2 is crucial for the assembly of functional KATP channels.

Reduced effectiveness of HMR 1098 in blocking cardiac sarcolemmal KATP channels during metabolic stress

ATP-sensitive K(+) (K(ATP)) channels are involved in ischemic cardioprotection induced by preconditioning (IPC), though the relative role of sarcolemmal (sK(ATP)) and mitochondrial (mitoK(ATP)) channels remains controversial. The sK(ATP)-selective sulphonylthiourea HMR 1098 has often been reported to be without effect on ischemic cardioprotection, suggesting minimal involvement of sK(ATP). Since some sulphonylureas show reduced potency under conditions of metabolic stress, we used patch clamp to assess the ability of HMR 1098 to block sK(ATP) currents of adult rat ventricular myocytes activated by metabolic inhibition (MI, NaCN+iodoacetate). In contrast to the prototype sulphonylurea glibenclamide, HMR 1098 (10 muM) was without effect on sK(ATP) currents, and also did not inhibit MI-induced action potential shortening. However, HMR 1098 blocked sK(ATP) current induced by the K(ATP) opener pinacidil (IC(50)=0.36+/-0.02 muM), and reversed pinacidil-induced action potential shortening. In inside-out patches, block by HMR 1098 was relieved by increasing MgADP concentrations (1-100 muM). HMR 1098 inhibited pinacidil-activated recombinant Kir6.2/SUR2A channels with a similar IC(50) (0.30+/-0.04 muM), but was less effective when channels were activated by low intracellular ATP. HMR 1098 displaced binding of the pinacidil analogue [(3)H]P1075 to native cardiac membranes with a biphasic inhibition curve. Our results show that HMR 1098 becomes a much less effective inhibitor of sK(ATP) during metabolic stress, and suggest that the lack of effect of HMR 1098 on ischemic cardioprotection reported in some studies may represent loss of block by the drug under these conditions rather than a lack of involvement of sK(ATP) channels.

Principal role of adenylyl cyclase 6 in K+ channel regulation and vasodilator signalling in vascular smooth muscle cells

Aims Membrane potential is a key determinant of vascular tone and many vasodilators act through the modulation of ion channel currents [e.g. the ATP-sensitive potassium channel (KATP)] involved in setting the membrane potential. Adenylyl cyclase (AC) isoenzymes are potentially important intermediaries in such vasodilator signalling pathways. Vascular smooth muscle cells (VSMCs) express multiple AC isoenzymes, but the reason for such redundancy is unknown. We investigated which of these isoenzymes are involved in vasodilator signalling and regulation of vascular ion channels important in modulating membrane potential. Methods and results AC isoenzymes were selectively depleted (by >75%) by transfection of cultured VSMCs with selective short interfering RNA sequences. AC6 was the predominant isoenzyme involved in vasodilator-mediated cAMP accumulation in VSMCs, accounting for ∼60% of the total response to β-adrenoceptor (β-AR) stimulation. AC3 played a minor role in β-AR signalling, whereas AC5 made no significant contribution. AC6 was also the principal isoenzyme involved in β-AR-mediated protein kinase A (PKA) signalling (determined using the fluorescent biosensor for PKA activity, AKAR3) and the substantial β-AR/PKA-dependent enhancement of KATP current. KATP current was shown to play a vital role in setting the resting membrane potential and in mediating the hyperpolarization observed upon β-AR stimulation. Conclusion AC6, but not the closely related AC5, plays a principal role in vasodilator signalling and regulation of the membrane potential in VSMCs. These findings identify AC6 as a vital component in the vasodilatory apparatus central to the control of blood pressure.

Effect of metabolic inhibition on glimepiride block of native and cloned cardiac sarcolemmal KATP channels

We have investigated the effects of the sulphonylurea, glimepiride, currently used to treat type 2 diabetes, on ATP‐sensitive K+ (KATP) currents of rat cardiac myocytes and on their cloned constituents Kir6.2 and SUR2A expressed in HEK 293 cells. Glimepiride blocked pinacidil‐activated whole‐cell KATP currents of cardiac myocytes with an IC50 of 6.8 nM, comparable to the potency of glibenclamide in these cells. Glimepiride blocked KATP channels formed by co‐expression of Kir6.2/SUR2A subunits in HEK 293 cells in outside‐out excised patches with a similar IC50 of 6.2 nM. Glimepiride was much less effective at blocking KATP currents activated by either metabolic inhibition (MI) with CN− and iodoacetate or by the KATP channel opener diazoxide in the presence of inhibitors of F0/F1‐ATPase (oligomycin) and creatine kinase (DNFB). Thus 10 μM glimepiride blocked pinacidil‐activated currents by >99%, MI‐activated currents by 70% and diazoxide‐activated currents by 82%. In inside‐out patches from HEK 293 cells expressing the cloned KATP channel subunits Kir6.2/SUR2A, increasing the concentration of ADP (1 – 100 μM), in the presence of 100 nM glimepiride, lead to significant increases in Kir6.2/SUR2A channel activity. However, over the range tested, ADP did not affect cloned KATP channel activity in the presence of 100 nM glibenclamide. These results are consistent with the suggestion that ADP reduces glimepiride block of KATP channels. Our results show that glimepiride is a potent blocker of native cardiac KATP channels activated by pinacidil and blocks cloned Kir6.2/SUR2A channels activated by ATP depletion with similar potency. However, glimepiride is much less effective when KATP channels are activated by MI and this may reflect a reduction in glimepiride block by increased intracellular ADP.