Mutations in KCNJ2, the gene encoding the human inward rectifier potassium channel Kir2.1, have been identified in Andersen syndrome (or Andersen‐Tawil syndrome), an inherited disorder characterized ...by periodic paralysis, cardiac arrhythmias, and dysmorphic features. We identified and characterized two novel KCNJ2 mutations (c.220A>G/p.T74A and c.443G>C/p.G144A) associated with Andersen syndrome. Heterologous expression of a recombinant wild type human KCNJ2 cDNA (WT‐KCNJ2) in HEK‐293 cells results in robust inward rectifying currents, but we did not observe measurable currents from cells expressing either mutant. Cells co‐transfected with WT‐KCNJ2 and either mutant exhibited substantially lower whole‐cell current amplitude consistent with a dominant‐negative suppression of WT‐KCNJ2 by the mutant channels. Both p.T74A and p.G144A exhibit robust plasma membrane expression, but a third previously reported allele (p.C101R) exhibited impaired trafficking. Our results demonstrate functional consequences of two novel trafficking‐competent KCNJ2 mutations associated with Andersen syndrome and expand our knowledge of allelic diversity in this disease. Published 2006 Wiley‐Liss, Inc.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
We sought to identify the electrophysiologic basis of life-threatening events associated with polymorphic ventricular tachycardia (PVT) in young patients with heterozygous KCNJ2 mutations. PVT ...describes a beat-to-beat alternating QRS axis and morphology during ventricular tachycardia. PVT may be well tolerated and even asymptomatic in young patients without other heart disease, but an association with syncope, cardiac arrest, or sudden death has long been known. Little is known of the basis of life-threatening events associated with PVT in this setting. We identified heterozygous KCNJ2 mutations (R67W and C101R respectively) in 2 adolescents with PVT (cycle length > 375 ms, < 160 beats/minute). Biophysical properties of wild-type and mutant KCNJ2 channels were characterized during heterologous expression in Xenopus oocytes. Despite a large tachycardia burden, neither patient experienced symptoms during electrocardiographic documentation of PVT. One patient had a history of cardiac arrest, but neither had other evidence of heart disease. Both patients were treated with an implantable cardioverter-defibrillator (ICD). In one patient, ICD interrogation identified rapid ventricular tachycardia (cycle length of 190 to 270 ms), terminated with a single 29-J asynchronous shock, as the cause of 2 syncopal episodes occurring 19 months apart. Biophysical characterization of KCNJ2-C101R demonstrated a loss-of-function and a dominant-negative effect on Kir2.1. Similar effects were previously observed for KCNJ2-R67W. Heterozygous mutations in KCNJ2 can cause life-threatening ventricular arrhythmias. Arrhythmia documented during cardiac arrest is rapid ventricular tachycardia; ICD is effective therapy for cardiac arrest in patients with PVT due to KCNJ2 mutation.
Voltage-gated sodium channel (NaV) mutations cause genetic pain disorders that range from severe paroxysmal pain to a congenital inability to sense pain. Previous studies on NaV1.7 and NaV1.8 ...established clear relationships between perturbations in channel function and divergent clinical phenotypes. By contrast, studies of NaV1.9 mutations have not revealed a clear relationship of channel dysfunction with the associated and contrasting clinical phenotypes. Here, we have elucidated the functional consequences of a NaV1.9 mutation (L1302F) that is associated with insensitivity to pain. We investigated the effects of L1302F and a previously reported mutation (L811P) on neuronal excitability. In transfected heterologous cells, the L1302F mutation caused a large hyperpolarizing shift in the voltage-dependence of activation, leading to substantially enhanced overlap between activation and steady-state inactivation relationships. In transfected small rat dorsal root ganglion neurons, expression of L1302F and L811P evoked large depolarizations of the resting membrane potential and impaired action potential generation. Therefore, our findings implicate a cellular loss of function as the basis for impaired pain sensation. We further demonstrated that a U-shaped relationship between the resting potential and the neuronal action potential threshold explains why NaV1.9 mutations that evoke small degrees of membrane depolarization cause hyperexcitability and familial episodic pain disorder or painful neuropathy, while mutations evoking larger membrane depolarizations cause hypoexcitability and insensitivity to pain.
Voltage-gated sodium channel (Na.sub.v) mutations cause genetic pain disorders that range from severe paroxysmal pain to a congenital inability to sense pain. Previous studies on Na.sub.v1.7 and ...Na.sub.v1.8 established clear relationships between perturbations in channel function and divergent clinical phenotypes. By contrast, studies of Na.sub.v1.9 mutations have not revealed a clear relationship of channel dysfunction with the associated and contrasting clinical phenotypes. Here, we have elucidated the functional consequences of a Na.sub.v1.9 mutation (L1302F) that is associated with insensitivity to pain. We investigated the effects of L1302F and a previously reported mutation (L811P) on neuronal excitability. In transfected heterologous cells, the L1302F mutation caused a large hyperpolarizing shift in the voltage-dependence of activation, leading to substantially enhanced overlap between activation and steady-state inactivation relationships. In transfected small rat dorsal root ganglion neurons, expression of L1302F and L811P evoked large depolarizations of the resting membrane potential and impaired action potential generation. Therefore, our findings implicate a cellular loss of function as the basis for impaired pain sensation. We further demonstrated that a U-shaped relationship between the resting potential and the neuronal action potential threshold explains why Na.sub.v1.9 mutations that evoke small degrees of membrane depolarization cause hyperexcitability and familial episodic pain disorder or painful neuropathy, while mutations evoking larger membrane depolarizations cause hypoexcitability and insensitivity to pain.
PURPOSE OF REVIEWRecent findings relevant to the renal ClC chloride channels/transporters are reviewed with a focus on structure–function relationships, regulation of trafficking, role in blood ...pressure control, and pharmacology.
RECENT FINDINGSThe ClC proteins include plasma membrane Cl channels and vesicular Cl/H exchangers. Recent experiments have revealed further details regarding the structure and mechanism of the permeation path. X-ray crystallographic and electrophysiological studies have identified two glutamate residues required for gated Cl movement and proton permeation in bacterial and two mammalian (ClC-4, ClC-5) ClC transporters. In renal ClC channels (ClC-Ka, ClC-Kb), both glutamate residues are replaced by valine, leading to speculation about critical differences between transporter and channel members of the ClC family. New information about the physiological regulation of renal ClC proteins has implicated the Nedd4 ubiquitin ligases and serum and glucocorticoid-inducible kinases in controlling functional levels of ClC-5 and ClC-K/barttin in renal cells.
SUMMARYClC proteins are critical for many clinically relevant physiological events. New insights into fundamental structure–function relationships, mechanisms of ion translocation, cellular regulation, and roles in human disease have increased attention on ClC proteins as important potential therapeutic targets.
P-glycoprotein is a membrane ATPase that transports drugs out of cells and confers resistance to a variety of chemically unrelated drugs (multidrug resistance). P-glycoprotein is phosphorylated by ...protein kinase C (PKC), and PKC blockers reduce P-glycoprotein phosphorylation and increase drug accumulation. These observations suggest that phosphorylation of P-glycoprotein stimulates drug transport. However, there is evidence that PKC inhibitors directly interact with P-glycoprotein, and therefore the mechanism of their effects on P-glycoprotein-mediated drug transport and the possible role of phosphorylation in the regulation of P-glycoprotein function remain unclear. In the present work, we studied the effects of different kinds of PKC inhibitors on drug transport in cells expressing wild-type human P-glycoprotein and a PKC phosphorylation-defective mutant. We demonstrated that PKC blockers inhibit drug transport by mechanisms independent of P-glycoprotein phosphorylation. Inhibition by the blockers occurs by (i) direct competition with transported drugs for binding to P-glycoprotein, and (ii) indirect inhibition through a pathway that involves PKC inhibition, but is independent of P-glycoprotein phosphorylation. The effects of the blockers on P-glycoprotein phosphorylation do not seem to play an important role, but the PKC-signaling pathway regulates P-glycoprotein-mediated drug transport.
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IJS, IMTLJ, KILJ, KISLJ, NUK, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
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