Available evidence indicates voltage-gated Na
channels (VGSCs) in peripheral sensory neurons are essential for the pain and hypersensitivity associated with tissue injury. However, our understanding ...of the biophysical and pharmacological properties of the channels in sensory neurons is largely based on the study of heterologous systems or rodent tissue, despite evidence that both expression systems and species differences influence these properties. Therefore, we sought to determine the extent to which the biophysical and pharmacological properties of VGSCs were comparable in rat and human sensory neurons. Whole cell patch clamp techniques were used to study Na
currents in acutely dissociated neurons from human and rat. Our results indicate that while the two major current types, generally referred to as tetrodotoxin (TTX)-sensitive and TTX-resistant were qualitatively similar in neurons from rats and humans, there were several differences that have important implications for drug development as well as our understanding of pain mechanisms.
Voltage-gated sodium (Na V 1) channels play a critical role in modulating the excitability of sensory neurons, and human genetic evidence points to Na V 1.7 as an essential contributor to pain ...signaling. Human loss-of-function mutations in SCN9A, the gene encoding Na V 1.7, cause channelopathy-associated indifference to pain (CIP), whereas gain-of-function mutations are associated with two
inherited painful neuropathies. Although the human genetic data make Na V 1.7 an attractive target for the development of analgesics, pharmacological proof-of-concept in experimental pain models requires
Na V 1.7-selective channel blockers. Here, we show that the tarantula venom peptide ProTx-II selectively interacts with Na V 1.7 channels, inhibiting Na V 1.7 with an IC 50 value of 0.3 nM, compared with IC 50 values of 30 to 150 nM for other heterologously expressed Na V 1 subtypes. This subtype selectivity was abolished by a point mutation in DIIS3. It is interesting that application of ProTx-II
to desheathed cutaneous nerves completely blocked the C-fiber compound action potential at concentrations that had little
effect on Aβ-fiber conduction. ProTx-II application had little effect on action potential propagation of the intact nerve,
which may explain why ProTx-II was not efficacious in rodent models of acute and inflammatory pain. Mono-iodo-ProTx-II ( 125 I-ProTx-II) binds with high affinity ( K d = 0.3 nM) to recombinant hNa V 1.7 channels. Binding of 125 I-ProTx-II is insensitive to the presence of other well characterized Na V 1 channel modulators, suggesting that ProTx-II binds to a novel site, which may be more conducive to conferring subtype selectivity
than the site occupied by traditional local anesthetics and anticonvulsants. Thus, the 125 I-ProTx-II binding assay, described here, offers a new tool in the search for novel Na V 1.7-selective blockers.
Cardiac ion channels Priest, Birgit T; McDermott, Jeff S
Channels,
11/2015, Letnik:
9, Številka:
6
Journal Article
Recenzirano
Odprti dostop
Ion channels are critical for all aspects of cardiac function, including rhythmicity and contractility. Consequently, ion channels are key targets for therapeutics aimed at cardiac pathophysiologies ...such as atrial fibrillation or angina. At the same time, off-target interactions of drugs with cardiac ion channels can be the cause of unwanted side effects. This manuscript aims to review the physiology and pharmacology of key cardiac ion channels. The intent is to highlight recent developments for therapeutic development, as well as elucidate potential mechanisms for drug-induced cardiac side effects, rather than present an in-depth review of each channel subtype.
Lipoprotein(a) (Lp(a)), an independent, causal cardiovascular risk factor, is a lipoprotein particle that is formed by the interaction of a low-density lipoprotein (LDL) particle and ...apolipoprotein(a) (apo(a))
. Apo(a) first binds to lysine residues of apolipoprotein B-100 (apoB-100) on LDL through the Kringle IV (K
) 7 and 8 domains, before a disulfide bond forms between apo(a) and apoB-100 to create Lp(a) (refs.
). Here we show that the first step of Lp(a) formation can be inhibited through small-molecule interactions with apo(a) K
7-8. We identify compounds that bind to apo(a) K
7-8, and, through chemical optimization and further application of multivalency, we create compounds with subnanomolar potency that inhibit the formation of Lp(a). Oral doses of prototype compounds and a potent, multivalent disruptor, LY3473329 (muvalaplin), reduced the levels of Lp(a) in transgenic mice and in cynomolgus monkeys. Although multivalent molecules bind to the Kringle domains of rat plasminogen and reduce plasmin activity, species-selective differences in plasminogen sequences suggest that inhibitor molecules will reduce the levels of Lp(a), but not those of plasminogen, in humans. These data support the clinical development of LY3473329-which is already in phase 2 studies-as a potent and specific orally administered agent for reducing the levels of Lp(a).
The transmission of pain signals after injury or inflammation depends in part on increased excitability of primary sensory neurons. Nociceptive neurons express multiple subtypes of voltage-gated ...sodium channels ( NaV1 s), each of which possesses unique features that may influence primary afferent excitability. Here, we examined the contribution of NaV1.9 to nociceptive signaling by studying the electrophysiological and behavioral phenotypes of mice with a disruption of the SCN11A gene, which encodes NaV1.9. Our results confirm that NaV1.9 underlies the persistent tetrodotoxin-resistant current in small-diameter dorsal root ganglion neurons but suggest that this current contributes little to mechanical thermal responsiveness in the absence of injury or to mechanical hypersensitivity after nerve injury or inflammation. However, the expression of NaV1.9 contributes to the persistent thermal hypersensitivity and spontaneous pain behavior after peripheral inflammation. These results suggest that inflammatory mediators modify the function of NaV1.9 to maintain inflammation-induced hyperalgesia.
Kir1.1 (renal outer medullary K+) channels are potassium channels expressed almost exclusively in the kidney and play a role in the body's electrolyte and water balance. Potassium efflux through ...Kir1.1 compliments the role of transporters and sodium channels that are the targets of known diuretics. Consequently, loss-of-function mutations in men and rodents are associated with salt wasting and low blood pressure. On this basis, Kir1.1 inhibitors may have value in the treatment of hypertension and heart failure. Efforts to develop small molecule Kir1.1 inhibitors produced MK-7145, which entered into clinical trials. The present manuscript describes the structure-activity relationships associated with this scaffold alongside other preclinical Kir1.1 blockers.
Resurgent sodium currents contribute to the regeneration of action potentials and enhanced neuronal excitability. Tetrodotoxin-sensitive (TTX-S) resurgent currents have been described in many ...different neuron populations, including cerebellar and dorsal root ganglia (DRG) neurons. In most cases, sodium channel Nav1.6 is the major contributor to these TTX-S resurgent currents. Here we report a novel TTX-resistant (TTX-R) resurgent current recorded from rat DRG neurons. The TTX-R resurgent currents are similar to classic TTX-S resurgent currents in many respects, but not all. As with TTX-S resurgent currents, they are activated by membrane repolarization, inhibited by lidocaine, and enhanced by a peptide-mimetic of the β4 sodium channel subunit intracellular domain. However, the TTX-R resurgent currents exhibit much slower kinetics, occur at more depolarized voltages, and are sensitive to the Nav1.8 blocker A803467. Moreover, coimmunoprecipitation experiments from rat DRG lysates indicate the endogenous sodium channel β4 subunits associate with Nav1.8 in DRG neurons. These results suggest that slow TTX-R resurgent currents in DRG neurons are mediated by Nav1.8 and are generated by the same mechanism underlying TTX-S resurgent currents. We also show that both TTX-S and TTX-R resurgent currents in DRG neurons are enhanced by inflammatory mediators. Furthermore, the β4 peptide increased excitability of small DRG neurons in the presence of TTX. We propose that these slow TTX-R resurgent currents contribute to the membrane excitability of nociceptive DRG neurons under normal conditions and that enhancement of both types of resurgent currents by inflammatory mediators could contribute to sensory neuronal hyperexcitability associated with inflammatory pain.
Blockers of the Delayed-Rectifier Potassium Current in Pancreatic β-Cells Enhance Glucose-Dependent Insulin Secretion
James Herrington 1 ,
Yun-Ping Zhou 2 ,
Randal M. Bugianesi 1 ,
Paula M. Dulski 1 ...,
Yue Feng 2 ,
Vivien A. Warren 1 ,
McHardy M. Smith 1 ,
Martin G. Kohler 1 ,
Victor M. Garsky 3 ,
Manuel Sanchez 4 ,
Michael Wagner 1 ,
Kristin Raphaelli 1 ,
Priya Banerjee 1 ,
Chinweze Ahaghotu 1 ,
Denise Wunderler 1 ,
Birgit T. Priest 1 ,
John T. Mehl 5 ,
Maria L. Garcia 1 ,
Owen B. McManus 1 ,
Gregory J. Kaczorowski 1 and
Robert S. Slaughter 1
1 Department of Ion Channels, Merck Research Laboratories, Rahway, New Jersey
2 Department of Metabolic Disorders–Diabetes, Merck Research Laboratories, Rahway, New Jersey
3 Department of Medicinal Chemistry, Merck Research Laboratories, West Point, Pennsylvania
4 Department of Farmacologia, Universidad de Oviedo, Oviedo, Spain
5 Department of Drug Metabolism, Merck Research Laboratories, West Point, Pennsylvania
Address correspondence and reprint requests to James Herrington, Merck Research Laboratories, RY80N-C31, P.O. Box 2000, Rahway,
NJ 07065-0900. E-mail: james_herrington{at}merck.com
Abstract
Delayed-rectifier K + currents (I DR ) in pancreatic β-cells are thought to contribute to action potential repolarization and thereby modulate insulin secretion.
The voltage-gated K + channel, K V 2.1, is expressed in β-cells, and the biophysical characteristics of heterologously expressed channels are similar to those
of I DR in rodent β-cells. A novel peptidyl inhibitor of K V 2.1/K V 2.2 channels, guangxitoxin (GxTX)-1 (half-maximal concentration ∼1 nmol/l), has been purified, characterized, and used to
probe the contribution of these channels to β-cell physiology. In mouse β-cells, GxTX-1 inhibits 90% of I DR and, as for K V 2.1, shifts the voltage dependence of channel activation to more depolarized potentials, a characteristic of gating-modifier
peptides. GxTX-1 broadens the β-cell action potential, enhances glucose-stimulated intracellular calcium oscillations, and
enhances insulin secretion from mouse pancreatic islets in a glucose-dependent manner. These data point to a mechanism for
specific enhancement of glucose-dependent insulin secretion by applying blockers of the β-cell I DR , which may provide advantages over currently used therapies for the treatment of type 2 diabetes.
GSIS, glucose-stimulated insulin secretion
GxTX, guangxitoxin
HaTX, hanatoxin
HPLC, high-performance liquid chromatography
IDR, delayed-rectifier K+ currents
Katp channel, ATP-sensitive K+ channel
KRB, Krebs-Ringer bicarbonate
TFA, trifluoroacetic acid
Footnotes
J.H. and Y.-P.Z. contributed equally to this article.
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore
be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Accepted December 21, 2005.
Received June 21, 2005.
DIABETES
•PKC activation increased resurgent currents produced by hNav1.7.•Phosphorylation-mimicking mutations at S1479 increased resurgent currents.•Phosphorylation-deficient mutation prevented increasing ...effects of PKC.•These data suggest PKC increases resurgent currents through S1479 phosphorylation.
Resurgent sodium currents likely play a role in modulating neuronal excitability. Here we studied whether protein kinase C (PKC) activation can increase resurgent currents produced by the human sodium channel hNav1.7. We found that a PKC agonist significantly enhanced hNav1.7-mediated resurgent currents and this was prevented by PKC antagonists. The enhancing effects were replicated by two phosphorylation-mimicking mutations and were prevented by a phosphorylation-deficient mutation at a conserved PKC phosphorylation site (Serine 1479). Our results suggest that PKC can increase sodium resurgent currents through phosphorylation of a conserved Serine residue located in the domain III–IV linker of sodium channels.
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