Robert J. French

Protein Kinase C–Induced Changes in the Stoichiometry of ATP Binding Activate Cardiac ATP-Sensitive K+ Channels A Possible Mechanistic Link to Ischemic Preconditioning

Activation of both ATP-sensitive K+ (KATP) channels and the enzyme protein kinase C (PKC) has been associated with the cardioprotective response of ischemic preconditioning. We recently showed that at low cytoplasmic ATP (< or = 50 mumol/L), PKC inhibits KATP channel activity. This finding is surprising, as both KATP channels and PKC are activated during preconditioning. However, PKC also altered ATP binding to the channel, changing the Hill coefficient from approximately 2 to approximately 1. This apparent change in stoichiometry would lead to a PKC-induced activation of KATP channels at more physiological (millimolar) levels of ATP. The aim of the present study was to determine whether PKC activates cardiac KATP channels at millimolar levels of ATP. The effects of PKC on single KATP channels were studied at millimolar internal ATP levels using excised inside-out membrane patches from rabbit ventricular myocytes. Application of purified constitutively active PKC (20 nmol/L) to the intracellular surface of the patches produced an approximately threefold increase in the channel open probability. The specific PKC inhibitor peptide PKC(19-31) prevented this increase. Heat-inactivated PKC had no effect on KATP channel properties. KATP channel activity spontaneously returned to control levels after washout of PKC. This spontaneous reversal did not occur in the presence of 5 nmol/L okadaic acid, suggesting that the reversal of PKCs action is dependent on activity of a membrane-associated type 2A protein phosphatase (PP2A). In the presence of exogenous PP2A (7.5 nmol/L), PKC had no effect. We conclude that the PKC-induced increase in KATP channel activity at millimolar ATP results from a crossing of the ATP concentration-response curves for inhibition of the phosphorylated and nonphosphorylated forms of the channel. This identifies a mechanism by which PKC activates KATP channels at near physiological levels of ATP and thus could link these two components in a signaling pathway that induces ischemic preconditioning.

Blockage of squid axon potassium conductance by internal tetra-N-alkylammonium ions of various sizes

We have studied the effects of the tetra-n-alkylammonium (TAA) ions, (CnH2n+1)4N+, n = 1-6, on the potassium conductance of voltage-clamped squid giant axons. Studies using tetrahexylammonium were not quantitatively analyzed as its effect was insufficiently reversible. Each in this series of symmetric ions of graded size blocks the potassium conductance when added to the internal perfusion fluid. There is a general trend for blocking potency to increase with increasing size. We attribute this to stronger interactions of the longer alkyl side chains with hydrophobic regions of the membrane near the channels. Steady-state block by the TAA ions, n = 2-5, showed identical voltage dependence, apparently sensing about 15% of the transmembrane voltage, and kinetics block onset were qualitatively similar. We conclude that the site of action for these ions is the same. Block by TMA is about twice as steeply dependent on voltage. In its action, TMA resembles the alkali cations (French et al., 1979, Biophys, J. 25(2, pt. 2):307a) more than the larger TAA ions. Our results suggest that access to the inner mouth of the K channel is even less restricted than has been previously thought. A calculation indicates that the lumen of the channel cannot be both wide enough to admit the TAA ions and long enough to account for the voltage dependence of block. We consider possible ways to resolve this paradox.

Targeted polyphosphatase expression alters mitochondrial metabolism and inhibits calcium-dependent cell death

Polyphosphate (polyP) consists of tens to hundreds of phosphates, linked by ATP-like high-energy bonds. Although polyP is present in mammalian mitochondria, its physiological roles there are obscure. Here, we examine the involvement of polyP in mitochondrial energy metabolism and ion transport. We constructed a vector to express a mitochondrially targeted polyphosphatase, along with a GFP fluorescent tag. Specific reduction of mitochondrial polyP, by polyphosphatase expression, significantly modulates mitochondrial bioenergetics, as indicated by the reduction of inner membrane potential and increased NADH levels. Furthermore, reduction of polyP levels increases mitochondrial capacity to accumulate calcium and reduces the likelihood of the calcium-induced mitochondrial permeability transition, a central event in many types of necrotic cell death. This confers protection against cell death, including that induced by β-amyloid peptide, a pathogenic agent in Alzheimers disease. These results demonstrate a crucial role played by polyP in mitochondrial function of mammalian cells.

Optimizing planar lipid bilayer single-channel recordings for high resolution with rapid voltage steps.

We describe two enhancements of the planar bilayer recording method which enable low-noise recordings of single-channel currents activated by voltage steps in planar bilayers formed on apertures in partitions separating two open chambers. First, we have refined a simple and effective procedure for making small bilayer apertures (25-80 micrograms diam) in plastic cups. These apertures combine the favorable properties of very thin edges, good mechanical strength, and low stray capacitance. In addition to enabling formation of small, low-capacitance bilayers, this aperture design also minimizes the access resistance to the bilayer, thereby improving the low-noise performance. Second, we have used a patch-clamp headstage modified to provide logic-controlled switching between a high-gain (50 G omega) feedback resistor for high-resolution recording and a low-gain (50 M omega) feedback resistor for rapid charging of the bilayer capacitance. The gain is switched from high to low before a voltage step and then back to high gain 25 microseconds after the step. With digital subtraction of the residual currents produced by the gain switching and electrostrictive changes in bilayer capacitance, we can achieve a steady current baseline within 1 ms after the voltage step. These enhancements broaden the range of experimental applications for the planar bilayer method by combining the high resolution previously attained only with small bilayers formed on pipette tips with the flexibility of experimental design possible with planar bilayers in open chambers. We illustrate application of these methods with recordings of the voltage-step activation of a voltage-gated potassium channel.

Structure/Function Characterization of μ-Conotoxin KIIIA, an Analgesic, Nearly Irreversible Blocker of Mammalian Neuronal Sodium Channels

Peptide neurotoxins from cone snails continue to supply compounds with therapeutic potential. Although several analgesic conotoxins have already reached human clinical trials, a continuing need exists for the discovery and development of novel non-opioid analgesics, such as subtype-selective sodium channel blockers. μ-Conotoxin KIIIA is representative of μ-conopeptides previously characterized as inhibitors of tetrodotoxin (TTX)-resistant sodium channels in amphibian dorsal root ganglion neurons. Here, we show that KIIIA has potent analgesic activity in the mouse pain model. Surprisingly, KIIIA was found to block most (>80%) of the TTX-sensitive, but only ∼20% of the TTX-resistant, sodium current in mouse dorsal root ganglion neurons. KIIIA was tested on cloned mammalian channels expressed in Xenopus oocytes. Both NaV1.2 and NaV1.6 were strongly blocked; within experimental wash times of 40–60 min, block was reversed very little for NaV1.2 and only partially for NaV1.6. Other isoforms were blocked reversibly: NaV1.3 (IC50 8 μm), NaV1.5 (IC50 284 μm), and NaV1.4 (IC50 80 nm). “Alanine-walk” and related analogs were synthesized and tested against both NaV1.2 and NaV1.4; replacement of Trp-8 resulted in reversible block of NaV1.2, whereas replacement of Lys-7, Trp-8, or Asp-11 yielded a more profound effect on the block of NaV1.4 than of NaV1.2. Taken together, these data suggest that KIIIA is an effective tool to study structure and function of NaV1.2 and that further engineering of μ-conopeptides belonging to the KIIIA group may provide subtype-selective pharmacological compounds for mammalian neuronal sodium channels and potential therapeutics for the treatment of pain.

Distinct myoprotective roles of cardiac sarcolemmal and mitochondrial KATP channels during metabolic inhibition and recovery

The protective roles of sarcolemmal (sarc) and mitochondrial (mito)KATP channels are un‐clear despite their apparent importance to ischemic preconditioning. We examined these roles by monitoring intracellular calcium ([Ca]int), using fura‐2 and fluo‐3, in enzymatically isolated rat right ventricular myocytes. Myocyte mortality, estimated using a trypan blue assay, changed approximately in parallel with changes in [Ca]int. Chemically induced hypoxia (CIH), induced by application of cyanide and 2‐deoxy‐glucose, caused a steady rise in [Ca]int. Calcium increased more rapidly on ‘reoxygenation’ by return to control solutions. The protein kinase C (PKC) activator PMA abolished both phases of calcium increase. The mitoKATP channel‐selective blocker 5‐hydroxydecanoate partially prevented the PMA‐induced protection during CIH, but not during reoxygenation. In contrast, HMR 1098, a sarcKATP channel‐selective blocker, abolished protection only during the reoxygenation. Adenosine (A1) receptor activation prevented or reduced increases in [Ca]int and improved cell viability via a PKC and mito/sarcKATP channel‐dependent mechanism. PKC‐dependent protection against cytoplasmic calcium increases was also observed in a human cell line (tsA201) transiently expressing sarcKATP channels. Protection was abolished only during the reoxygenation phase by the amino acid substitution (T180A) in the pore‐forming Kir6.2 subunit, a mutation previously shown to prevent PKC‐dependent modulation. Our data suggest that sarc and mitoKATP channel populations play distinct protective roles, triggered by PKC and/or adenosine, during chemically induced hypoxia/reoxygenation.—Light, P. E., Kanji, H. D., Manning Fox, J. E., French, R. J. Distinct myoprotective roles of cardiac sarcolem‐mal and mitochondrial KATP channels during metabolic inhibition and recovery. FASEB J. 15, 2586–2594 (2001)

Inorganic polyphosphate modulates TRPM8 channels

Polyphosphate (polyP) is an inorganic polymer built of tens to hundreds of phosphates, linked by high-energy phosphoanhydride bonds. PolyP forms complexes and modulates activities of many proteins including ion channels. Here we investigated the role of polyP in the function of the transient receptor potential melastatin 8 (TRPM8) channel. Using whole-cell patch-clamp and fluorescent calcium measurements we demonstrate that enzymatic breakdown of polyP by exopolyphosphatase (scPPX1) inhibits channel activity in human embryonic kidney and F-11 neuronal cells expressing TRPM8. We demonstrate that the TRPM8 channel protein is associated with polyP. Furthermore, addition of scPPX1 altered the voltage-dependence and blocked the activity of the purified TRPM8 channels reconstituted into planar lipid bilayers, where the activity of the channel was initiated by cold and menthol in the presence of phosphatidylinositol 4,5-biphosphate (PtdIns(4,5)P2). The biochemical analysis of the TRPM8 protein also uncovered the presence of poly-(R)-3-hydroxybutyrate (PHB), which is frequently associated with polyP. We conclude that the TRPM8 protein forms a stable complex with polyP and its presence is essential for normal channel activity.

Interactions between a pore-blocking peptide and the voltage sensor of the sodium channel: an electrostatic approach to channel geometry.

Few experimental data illuminate the relationship between the molecular structures that mediate ion conduction through voltage-dependent ion channels and the structures responsible for sensing transmembrane voltage and controlling gating. To fill this void, we have used a strongly cationic, mutated mu-conotoxin peptide, which only partially blocks current through voltage-dependent sodium channels, to study voltage-dependent activation gating in both bound and unbound channels. When the peptide binds to the ion-conducting pore, it inhibit channel opening, necessitating stronger depolarization for channel activation. We show that this activation shift could result entirely from electrostatic inhibition of the movement of the voltage-sensing S4 charges and estimate the approximate physical distance through which the S4 charges move.

Structure/function characterization of -conotoxin kiiia, an analgesic, nearly irreversible blocker of neuronal mammalian sodium channels

Peptide neurotoxins from cone snails continue to supply compounds with therapeutic potential. Although several analgesic conotoxins have already reached human clinical trials, a continuing need exists for the discovery and development of novel non-opioid analgesics, such as subtype-selective sodium channel blockers. μ-Conotoxin KIIIA is representative of μ-conopeptides previously characterized as inhibitors of tetrodotoxin (TTX)-resistant sodium channels in amphibian dorsal root ganglion neurons. Here, we show that KIIIA has potent analgesic activity in the mouse pain model. Surprisingly, KIIIA was found to block most (>80%) of the TTX-sensitive, but only ∼20% of the TTX-resistant, sodium current in mouse dorsal root ganglion neurons. KIIIA was tested on cloned mammalian channels expressed in Xenopus oocytes. Both NaV1.2 and NaV1.6 were strongly blocked; within experimental wash times of 40–60 min, block was reversed very little for NaV1.2 and only partially for NaV1.6. Other isoforms were blocked reversibly: NaV1.3 (IC50 8 μm), NaV1.5 (IC50 284 μm), and NaV1.4 (IC50 80 nm). “Alanine-walk” and related analogs were synthesized and tested against both NaV1.2 and NaV1.4; replacement of Trp-8 resulted in reversible block of NaV1.2, whereas replacement of Lys-7, Trp-8, or Asp-11 yielded a more profound effect on the block of NaV1.4 than of NaV1.2. Taken together, these data suggest that KIIIA is an effective tool to study structure and function of NaV1.2 and that further engineering of μ-conopeptides belonging to the KIIIA group may provide subtype-selective pharmacological compounds for mammalian neuronal sodium channels and potential therapeutics for the treatment of pain.

Voltage-dependent block by saxitoxin of sodium channels incorporated into planar lipid bilayers

We have previously studied single, voltage-dependent, saxitoxin-(STX) blockable sodium channels from rat brain in planar lipid bilayers, and found that channel block by STX was voltage-dependent. Here we describe the effect of voltage on the degree of block and on the kinetics of the blocking reaction. From their voltage dependence and kinetics, it was possible to distinguish single-channel current fluctuations due to blocking and unblocking of the channels by STX from those caused by intrinsic channel gating. The use of batrachotoxin (BTX) to inhibit sodium-channel inactivation allowed recordings of stationary fluctuations over extended periods of time. In a range of membrane potentials where the channels were open greater than 98% of the time, STX block was voltage-dependent, provided sufficient time was allowed to reach a steady state. Hyperpolarizing potentials favored block. Both association (blocking) and dissociation (unblocking) rate constants were voltage-dependent. The equilibrium dissociation constants computed from the association and dissociation rate constants for STX block were about the same as those determined from the steady-state fractional reduction in current. The steepness of the voltage dependence was consistent with the divalent toxin sensing 30-40% of the transmembrane potential.