N. B. Standen

Tetraethylammonium block of Slowpoke calcium-activated potassium channels expressed in Xenopus oocytes: Evidence for tetrameric channel formation

Unitary currents were recorded from insideout membrane patches pulled from Xenopus oocytes that had been injected with RNA transcribed from a cDNA encoding the Drosophila maxi-K channel (Slowpoke). Site-directed mutagenesis was used to make cDNAs encoding channel subunits with single amino acid substitutions (Y308V and C309P). The extracellular side of the patch was exposed to tetraethylammonium (TEA) in the pipette solution; unitary currents in the presence of TEA were compared with currents in the absence of TEA to compute the inhibition. Amplitude distributions were fit by β functions to estimate the blocking and unblocking rate constants. For wild-type channels, TEA blocked with an apparent Kd of 80 μM at 0 mV and sensed 0.18 of the membrane electric field; the voltage dependence lay entirely in the blocking rate constant. TEA blocked currents through C309P channels with a similar affinity to wild-type at 0 mV, but this was not voltage-dependent. Currents through Y308V channels were very insensitive to any block by TEA; the apparent Kd at 0 mV was 26 mM and the blockade sensed 0.18 of the electric field. Oocytes injected with a mixture of RNAs encoding wild-type and Y308V channels showed unitary currents of four discrete amplitudes in the presence of 3 mM TEA; at 40 mV these corresponded to inhibitions of approximately 80%, 55%, 25% and 10%. These values agreed well with those expected for inhibition by TEA of currents through channels containing 3, 2, 1 and 0 tyrosine residues at the channel mouth, assuming that a tyrosine residue from each of four subunits contributes equally to the binding of the TEA ion. This indicates that Slowpoke channels form as tetramers.

ATP-sensitive K+ channel activation by calcitonin gene-related peptide and protein kinase A in pig coronary arterial smooth muscle

1 We used patch clamp to study whole‐cell K+ currents activated by calcitonin gene‐related peptide (CGRP) in smooth muscle cells freshly dissociated from pig coronary arteries. 2 CGRP (50 nm) activated an inward current at −60 mV in symmetrical 140 mm K+ that was blocked by glibenclamide (10 μm), an inhibitor of ATP‐sensitive potassium (KATP) channels. CGRP‐induced currents were larger in cells dialysed with 0.1 mm ATP than with 3.0 mm ATP. 3 Forskolin (10 μm) activated a glibenclamide‐sensitive current, as did intracellular dialysis with cAMP (100 μm). The catalytic subunit of cAMP‐dependent protein kinase (protein kinase A, PKA), added to the pipette solution, activated equivalent currents in five out of twelve cells. 4 CGRP‐induced currents were reduced by the PKA inhibitors adenosine 3′,5′‐cyclic monophosphorothioate, RP‐isomer, triethylammonium salt (Rp‐cAMPS; 100 μm) and N‐[2‐((p‐bromocinnamyl)amino)ethyl]‐5‐isoquinolinesulphonamide dihydrochloride (H‐89; 1 μm), and abolished by inclusion of a PKA inhibitor peptide in the pipette solution. 5 The β‐adrenergic agonist isoprenaline (10 μm) also activated a glibenclamide‐sensitive K+ current. 6 CGRP‐induced currents were unaffected by the inhibitor of cGMP‐dependent protein kinase (PKG) KT5823 (1 μm). Sodium nitroprusside (10 μm) did not activate a glibenclamide‐sensitive current in cells held at −60 mV, but did activate an outward current at +60 mV that was abolished by KT5823, or by 100 nm iberiotoxin (an inhibitor of BKCa channels). 7 Our findings suggest that CGRP activates coronary KATP channels through a pathway that involves adenylyl cyclase and PKA, but not PKG.

The properties and distribution of inward rectifier potassium currents in pig coronary arterial smooth muscle.

1. Whole‐cell potassium currents were studied in single smooth muscle cells enzymatically isolated from pig coronary arteries. 2. In cells isolated from small diameter branches of the left anterior descending coronary artery (LAD), an inward rectifier potassium current (IK(IR)) was identified, which was inhibited by extracellular barium ions, suggesting the presence of inward rectifier potassium (KIR) channels. 3. The conductance for IK(IR) measured in 6, 12, 60 and 140 mM extracellular potassium was a function of membrane potential and the extracellular potassium concentration. 4. On hyperpolarization, IK(IR) activated along an exponential time course with a time constant that was voltage dependent. 5. Inward rectifier current was compared in cells isolated from coronary vessels taken from different points along the vascular tree. Current density was greater in cells isolated from small diameter coronary arteries; at ‐140 mV it was ‐20.5 +/‐ 4.4 pA pF‐1 (n = 23) in 4th order branches of the LAD, but ‐0.8 +/‐ 0.2 pA pF‐1 (n = 11) in the LAD itself. 6. In contrast to IK(IR), there was little effect of arterial diameter on the density of voltage‐dependent potassium current; densities at +30 mV were 12.8 +/‐ 1.3 pA pF‐1 (n = 19) in 4th order branches and 17.4 +/‐ 3.1 pA pF‐1 (n = 11) in the LAD. 7. We conclude that KIR channels are present in pig coronary arteries, and that they are expressed at a higher density in small diameter arteries. The presence of an enhanced IK(IR) may have functional consequences for the regulation of cell membrane potential and tone in small coronary arteries.

Activation of ATP‐dependent K+ channels by hypoxia in smooth muscle cells isolated from the pig coronary artery.

1. The perforated patch technique with amphotericin B was used to record whole‐cell currents activated by hypoxia in smooth muscle cells, isolated enzymatically from pig coronary arteries. 2. Superfusion with hypoxic solution (O2 partial pressure, 25‐40 mmHg) activated an inward current at ‐60 mV in 143 mM extracellular K+. The reversal potential of the current induced by hypoxia shifted with extracellular [K+] as expected for a K+ current, while its current‐voltage relation was consistent with the channels showing little voltage dependence. 3. The hypoxia‐induced current was inhibited by glibenclamide (10 microM), but was unaffected by charybdotoxin (50 nM). 4. In whole‐cell recordings at ‐60 mV in 143 mM K+ solution, openings of single channels passing a current close to ‐2 pA could sometimes be detected in normoxic solution. Openings became more frequent during the onset of the response to hypoxia, when several levels could be detected. Channels with a similar conductance were activated by hypoxia in cell‐attached patches. 5. Our results suggest that hypoxia activates ATP‐dependent K+ channels. We discuss possible mechanisms by which this activation may occur.

Angiotensin II inhibits rat arterial KATP channels by inhibiting steady‐state protein kinase A activity and activating protein kinase Ce

1 We used whole‐cell patch clamp to investigate steady‐state activation of ATP‐sensitive K+ channels (KATP) of rat arterial smooth muscle by protein kinase A (PKA) and the pathway by which angiotensin II (Ang II) inhibits these channels. 2 Rp‐cAMPS, an inhibitor of PKA, did not affect KATP currents activated by pinacidil when the intracellular solution contained 0.1 mM ATP. However, when ATP was increased to 1.0 mM, inhibition of PKA reduced KATP current, while the phosphatase inhibitor calyculin A caused a small increase in current. 3 Ang II (100 nM) inhibited KATP current activated by the K+ channel opener pinacidil. The degree of inhibition was greater with 1.0 mM than with 0.1 mM intracellular ATP. The effect of Ang II was abolished by the AT1 receptor antagonist losartan. 4 The inhibition of KATP currents by Ang II was abolished by a combination of PKA inhibitor peptide 5‐24 (5 μM) and PKC inhibitor peptide 19‐27 (100 μM), while either alone caused only partial block of the effect. 5 In the presence of PKA inhibitor peptide, the inhibitory effect of Ang II was unaffected by the PKC inhibitor Gö 6976, which is selective for Ca2+‐dependent isoforms of PKC, but was abolished by a selective peptide inhibitor of the translocation of the ε isoform of PKC. 6 Our results indicate that KATP channels are activated by steady‐state phosphorylation by PKA at normal intracellular ATP levels, and that Ang II inhibits the channels both through activation of PKCε and inhibition of PKA.

Caveolae Localize Protein Kinase A Signaling to Arterial ATP-Sensitive Potassium Channels

Arterial ATP-sensitive K+ (KATP) channels are critical regulators of vascular tone, forming a focal point for signaling by many vasoactive transmitters that alter smooth muscle contractility and so blood flow. Clinically, these channels form the target of antianginal and antihypertensive drugs, and their genetic disruption leads to hypertension and sudden cardiac death through coronary vasospasm. However, whereas the biochemical basis of KATP channel modulation is well-studied, little is known about the structural or spatial organization of the signaling pathways that converge on these channels. In this study, we use discontinuous sucrose density gradients and Western blot analysis to show that KATP channels localize with an upstream signaling partner, adenylyl cyclase, to smooth muscle membrane fractions containing caveolin, a protein found exclusively in cholesterol and sphingolipid-enriched membrane invaginations known as caveolae. Furthermore, we show that an antibody against the KATP pore-forming subunit, Kir6.1 co-immunoprecipitates caveolin from arterial homogenates, suggesting that Kir6.1 and caveolin exist together in a complex. To assess whether the colocalization of KATP channels and adenylyl cyclase to smooth muscle caveolae has functional significance, we disrupt caveolae with the cholesterol-depleting agent, methyl-&bgr;-cyclodextrin. This reduces the cAMP-dependent protein kinase A–sensitive component of whole-cell KATP current, indicating that the integrity of caveolae is important for adenylyl cyclase–mediated channel modulation. These results suggest that to be susceptible to protein kinase A–dependent activation, arterial KATP channels need to be localized in the same lipid compartment as adenylyl cyclase; the results also provide the first indication of the spatial organization of signaling pathways that regulate KATP channel activity.

Properties of BKCa Channels Formed by Bicistronic Expression of hSloα and β1–4 Subunits in HEK293 Cells

Large-conductance Ca2+-activated K+ (BKCa) channels are sensitive to both voltage and internal [Ca2+] and are found in many tissues. Their physiological roles range from causing relaxation of smooth muscle to regulating the frequency of action potential firing. There is considerable variation between different tissues in their Ca2+- and voltage-dependence. Much of this variation results from the association of the pore-forming α subunit (hSloα) with different β subunits leading to altered channel properties. Since hSloα alone produces functional BKCa channels, we have used a bicistronic expression method to ensure that both α and β subunits are expressed, with the β subunit being in excess. Using this method we have investigated the effect of four β subunits (β1 to β4) on cloned BKCa channels. The four β subunits were individually cloned into a vector that had hSloα cDNA inserted downstream of an internal ribosome entry site. The constructs were transiently transfected into HEK293 cells together with a construct that expresses green fluorescent protein, as a marker for transfection. Fluorescent cells expressed BKCa channels whose currents were recorded from inside-out or outside-out patches. The currents we measured using this expression system were similar to those expressed in Xenopus oocytes by Brenner et al. (Brenner, R., Jegla, T.J., Wickenden, A., Liu, Y., Aldrich, R.W. 2000. Cloning and functional expression of novel large-conductance calcium-activated potassium channel β subunits, hKCNMB3 and hKCNMB4. J. Biol. Chem.275:6453-6461.)

Angiotensin II Inhibition of ATP‐Sensitive K+ Currents in Rat Arterial Smooth Muscle Cells Through Protein Kinase C

1 The effects of the vasoconstrictor angiotensin II (Ang II) on whole‐cell ATP‐sensitive K+currents (Ik,atp) of smooth muscle cells isolated enzymatically from rat mesenteric arteries were investigated using the patch clamp technique. 2 Ang II, at a physiological concentration (100 nm), reduced Ik,atp activated by 0.1 mm internal ATP and 10 μm levcromakalim by 36.4 ± 2.3%. 3 The protein kinase C (PKC) activator 1‐oleoyl‐2‐acetyl‐sn‐glycerol (OAG, 1 μm) reduced Ik,atp by 44.1 ± 2.7%. GDPβS (1 mm), included in the pipette solution, abolished the inhibition by Ang II, while that by OAG was unaffected. 4 Pretreatment with the PKC inhibitors staurosporine (100 nm) or calphostin C (500 nm) prevented the Ang II‐induced inhibition of Ik,atp. 5 Ang II inhibition was unaffected by cell dialysis with PKA inhibitor peptide (5 μm), and the PKA inhibitor Rp‐cAMPS (100 μ) did not reduce Ik,atp. 6 Our results suggest that Ang II modulates Katp channels through activation of PKC but not through inhibition of PKA.

The KATP channel opener diazoxide protects cardiac myocytes during metabolic inhibition without causing mitochondrial depolarization or flavoprotein oxidation

The KATP channel opener diazoxide has been proposed to protect cardiac muscle against ischaemia by opening mitochondrial KATP channels to depolarize the mitochondrial membrane potential, ΔΨm. We have used the fluorescent dye TMRE to measure ΔΨm in adult rat freshly isolated cardiac myocytes exposed to diazoxide and metabolic inhibition. Diazoxide, at concentrations that are highly cardioprotective (100 or 200 μM), caused no detectable increase in TMRE fluorescence (n=27 cells). However, subsequent application of the protonophore FCCP, which should collapse ΔΨm, led to large increases in TMRE fluorescence (>300%). Metabolic inhibition (MI: 2 mM NaCN+1 mM iodoacetic acid (IAA) led to an immediate partial depolarization of ΔΨm, followed after a few minutes delay by complete depolarization which was correlated with rigor contracture. Removal of metabolic inhibition led to abrupt mitochondrial repolarization followed in many cells by hypercontracture, indicated by cell rounding and loss of striated appearance. Prior application of diazoxide (100 μM) reduced the number of cells that hypercontracted after metabolic inhibition from 63.7±4.7% to 24.2±1.8% (P<0.0001). 5‐hydroxydeanoate (100 μM) reduced the protection of diazoxide (46.8±2.7% cells hypercontracted, P<0.0001 vs diazoxide alone). Diazoxide caused no detectable change in flavoprotein autofluorescence (n=26 cells). Our results suggest that mitochondrial depolarization and flavoprotein oxidation are not inevitable consequences of diazoxide application in intact cardiac myocytes, and that they are also not essential components of the mechanism by which it causes protection.

The intrinsic gating of inward rectifier K+ channels expressed from the murine IRK1 gene depends on voltage, K+ and Mg2+

1. We describe the cloning of the inward rectifier K+ channel IRK1 from genomic DNA of mouse; the gene is intronless. 2. The IRK1 gene can be stably expressed in murine erythroleukaemia (MEL) cells. Such transfected cells show inward rectification under whole‐cell recording. 3. Channels encoded by the IRK1 gene have an intrinsic gating that depends on voltage and [K+]o. Rate constants are reduced e‐fold as the driving force on K+(V‐EK) is reduced by 24.1 mV. 4. Removal of intracellular Mg2+ permits brief outward currents under depolarization. The instantaneous current‐voltage relation may be fitted by an appropriate constant field expression. 5. Removal of intracellular Mg2+ speeds channel closure at positive voltages. In nominally zero [Mg2+]i, rate constants for the opening and closing of channels, processes which are first order, are similar to those of native channels.