Maria Virginia Soldovieri

Driving With No Brakes: Molecular Pathophysiology of Kv7 Potassium Channels

Kv7 potassium channels regulate excitability in neuronal, sensory, and muscular cells. Here, we describe their molecular architecture, physiological roles, and involvement in genetically determined channelopathies highlighting their relevance as targets for pharmacological treatment of several human disorders.

Genotype–phenotype correlations in neonatal epilepsies caused by mutations in the voltage sensor of Kv7.2 potassium channel subunits

Mutations in the KV7.2 gene encoding for voltage-dependent K+ channel subunits cause neonatal epilepsies with wide phenotypic heterogeneity. Two mutations affecting the same positively charged residue in the S4 domain of KV7.2 have been found in children affected with benign familial neonatal seizures (R213W mutation) or with neonatal epileptic encephalopathy with severe pharmacoresistant seizures and neurocognitive delay, suppression-burst pattern at EEG, and distinct neuroradiological features (R213Q mutation). To examine the molecular basis for this strikingly different phenotype, we studied the functional characteristics of mutant channels by using electrophysiological techniques, computational modeling, and homology modeling. Functional studies revealed that, in homomeric or heteromeric configuration with KV7.2 and/or KV7.3 subunits, both mutations markedly destabilized the open state, causing a dramatic decrease in channel voltage sensitivity. These functional changes were (i) more pronounced for channels incorporating R213Q- than R213W-carrying KV7.2 subunits; (ii) proportional to the number of mutant subunits incorporated; and (iii) fully restored by the neuronal Kv7 activator retigabine. Homology modeling confirmed a critical role for the R213 residue in stabilizing the activated voltage sensor configuration. Modeling experiments in CA1 hippocampal pyramidal cells revealed that both mutations increased cell firing frequency, with the R213Q mutation prompting more dramatic functional changes compared with the R213W mutation. These results suggest that the clinical disease severity may be related to the extent of the mutation-induced functional K+ channel impairment, and set the preclinical basis for the potential use of Kv7 openers as a targeted anticonvulsant therapy to improve developmental outcome in neonates with KV7.2 encephalopathy.

Decreased Subunit Stability as a Novel Mechanism for Potassium Current Impairment by a KCNQ2 C Terminus Mutation Causing Benign Familial Neonatal Convulsions

KCNQ2 and KCNQ3 K+ channel subunits underlie the muscarinic-regulated K+ current (IKM), a widespread regulator of neuronal excitability. Mutations in KCNQ2- or KCNQ3-encoding genes cause benign familiar neonatal convulsions (BFNCs), a rare autosomal-dominant idiopathic epilepsy of the newborn. In the present study, we have investigated, by means of electrophysiological, biochemical, and immunocytochemical techniques in transiently transfected cells, the consequences prompted by a BFNC-causing 1-bp deletion (2043ΔT) in the KCNQ2 gene; this frameshift mutation caused the substitution of the last 163 amino acids of the KCNQ2 C terminus and the extension of the subunit by additional 56 residues. The 2043ΔT mutation abolished voltage-gated K+ currents produced upon homomeric expression of KCNQ2 subunits, dramatically reduced the steady-state cellular levels of KCNQ2 subunits, and prevented their delivery to the plasma membrane. Metabolic labeling experiments revealed that mutant KCNQ2 subunits underwent faster degradation; 10-h treatment with the proteasomal inhibitor MG132 (20 μm) at least partially reversed such enhanced degradation. Co-expression with KCNQ3 subunits reduced the degradation rate of mutant KCNQ2 subunits and led to their expression on the plasma membrane. Finally, co-expression of KCNQ2 2043ΔT together with KCNQ3 subunits generated functional voltage-gated K+ currents having pharmacological and biophysical properties of heteromeric channels. Collectively, the present results suggest that mutation-induced reduced stability of KCNQ2 subunits may cause epilepsy in neonates.

Activation and desensitization of TRPV1 channels in sensory neurons by the PPARα agonist palmitoylethanolamide

Palmitoylethanolamide (PEA) is an endogenous fatty acid amide displaying anti‐inflammatory and analgesic actions. To investigate the molecular mechanism responsible for these effects, the ability of PEA and of pain‐inducing stimuli such as capsaicin (CAP) or bradykinin (BK) to influence intracellular calcium concentrations ([Ca2+]i) in peripheral sensory neurons, has been assessed in the present study. The potential involvement of the transcription factor PPARα and of TRPV1 channels in PEA‐induced effects was also studied.

A novel KCNQ2 K+channel mutation in benign neonatal convulsions and centrotemporal spikes

Patients with benign familial neonatal convulsions (BFNC) may develop various epilepsies or epilepsy-associated EEG traits. A heterozygous 1–base pair deletion (2043ΔT) in the KCNQ2 gene encoding for K+channel subunits was found in a patient with BFNC who showed centrotemporal spikes at age 3 years. Electrophysiologic studies showed that mutant K+channel subunits failed to give rise to functional homomeric channels or exert dominant-negative effects when expressed with KCNQ2/KCNQ3 subunits.

Early-onset epileptic encephalopathy caused by gain-of-function mutations in the voltage sensor of Kv7.2 and Kv7.3 potassium channel subunits.

Mutations in Kv7.2 (KCNQ2) and Kv7.3 (KCNQ3) genes, encoding for voltage-gated K+ channel subunits underlying the neuronal M-current, have been associated with a wide spectrum of early-onset epileptic disorders ranging from benign familial neonatal seizures to severe epileptic encephalopathies. The aim of the present work has been to investigate the molecular mechanisms of channel dysfunction caused by voltage-sensing domain mutations in Kv7.2 (R144Q, R201C, and R201H) or Kv7.3 (R230C) recently found in patients with epileptic encephalopathies and/or intellectual disability. Electrophysiological studies in mammalian cells transfected with human Kv7.2 and/or Kv7.3 cDNAs revealed that each of these four mutations stabilized the activated state of the channel, thereby producing gain-of-function effects, which are opposite to the loss-of-function effects produced by previously found mutations. Multistate structural modeling revealed that the R201 residue in Kv7.2, corresponding to R230 in Kv7.3, stabilized the resting and nearby voltage-sensing domain states by forming an intricate network of electrostatic interactions with neighboring negatively charged residues, a result also confirmed by disulfide trapping experiments. Using a realistic model of a feedforward inhibitory microcircuit in the hippocampal CA1 region, an increased excitability of pyramidal neurons was found upon incorporation of the experimentally defined parameters for mutant M-current, suggesting that changes in network interactions rather than in intrinsic cell properties may be responsible for the neuronal hyperexcitability by these gain-of-function mutations. Together, the present results suggest that gain-of-function mutations in Kv7.2/3 currents may cause human epilepsy with a severe clinical course, thus revealing a previously unexplored level of complexity in disease pathogenetic mechanisms.

The Ever Changing Moods of Calmodulin: How Structural Plasticity Entails Transductional Adaptability

The exceptional versatility of calmodulin (CaM) three-dimensional arrangement is reflected in the growing number of structural models of CaM/protein complexes currently available in the Protein Data Bank (PDB) database, revealing a great diversity of conformations, domain organization, and structural responses to Ca(2+). Understanding CaM binding is complicated by the diversity of target proteins sequences. Data mining of the structures shows that one face of each of the eight CaM helices can contribute to binding, with little overall difference between the Ca(2+) loaded N- and C-lobes and a clear prevalence of the C-lobe low Ca(2+) conditions. The structures reveal a remarkable variety of configurations where CaM binds its targets in a preferred orientation that can be reversed and where CaM rotates upon Ca(2+) binding, suggesting a highly dynamic metastable relation between CaM and its targets. Recent advances in structure-function studies and the discovery of CaM mutations being responsible for human diseases, besides expanding the role of CaM in human pathophysiology, are opening new exciting avenues for the understanding of the how CaM decodes Ca(2+)-dependent and Ca(2+)-independent signals.

Novel KCNQ2 and KCNQ3 Mutations in a Large Cohort of Families with Benign Neonatal Epilepsy: First Evidence for an Altered Channel Regulation by Syntaxin‐1A

Mutations in the KCNQ2 and KCNQ3 genes encoding for Kv7.2 (KCNQ2; Q2) and Kv7.3 (KCNQ3; Q3) voltage‐dependent K+ channel subunits, respectively, cause neonatal epilepsies with wide phenotypic heterogeneity. In addition to benign familial neonatal epilepsy (BFNE), KCNQ2 mutations have been recently found in families with one or more family members with a severe outcome, including drug‐resistant seizures with psychomotor retardation, electroencephalogram (EEG) suppression‐burst pattern (Ohtahara syndrome), and distinct neuroradiological features, a condition that was named “KCNQ2 encephalopathy.” In the present article, we describe clinical, genetic, and functional data from 17 patients/families whose electroclinical presentation was consistent with the diagnosis of BFNE. Sixteen different heterozygous mutations were found in KCNQ2, including 10 substitutions, three insertions/deletions and three large deletions. One substitution was found in KCNQ3. Most of these mutations were novel, except for four KCNQ2 substitutions that were shown to be recurrent. Electrophysiological studies in mammalian cells revealed that homomeric or heteromeric KCNQ2 and/or KCNQ3 channels carrying mutant subunits with newly found substitutions displayed reduced current densities. In addition, we describe, for the first time, that some mutations impair channel regulation by syntaxin‐1A, highlighting a novel pathogenetic mechanism for KCNQ2‐related epilepsies.

Atypical Gating Of M-Type Potassium Channels Conferred by Mutations in Uncharged Residues in the S4 Region of KCNQ2 Causing Benign Familial Neonatal Convulsions

Heteromeric assembly of KCNQ2 and KCNQ3 subunits underlie the M-current (IKM), a slowly activating and noninactivating neuronal K+ current. Mutations in KCNQ2 and KCNQ3 genes cause benign familial neonatal convulsions (BFNCs), a rare autosomal-dominant epilepsy of the newborn. In the present study, we describe the identification of a novel KCNQ2 heterozygous mutation (c587t) in a BFNC-affected family, leading to an alanine to valine substitution at amino acid position 196 located at the N-terminal end of the voltage-sensing S4 domain. The consequences on KCNQ2 subunit function prompted by the A196V substitution, as well as by the A196V/L197P mutation previously described in another BFNC-affected family, were investigated by macroscopic and single-channel current measurements in CHO cells transiently transfected with wild-type and mutant subunits. When compared with KCNQ2 channels, homomeric KCNQ2 A196V or A196V/L197P channels showed a 20 mV rightward shift in their activation voltage dependence, with no concomitant change in maximal open probability or single-channel conductance. Furthermore, current activation kinetics of KCNQ2 A196V channels displayed an unusual dependence on the conditioning prepulse voltage, being markedly slower when preceded by prepulses to more depolarized potentials. Heteromeric channels formed by KCNQ2 A196V and KCNQ3 subunits displayed gating changes similar to those of KCNQ2 A196V homomeric channels. Collectively, these results reveal a novel role for noncharged residues in the N-terminal end of S4 in controlling gating of IKM and suggest that gating changes caused by mutations at these residues may decrease IKM function, thus causing neuronal hyperexcitability, ultimately leading to neonatal convulsions.

EXPRESSION, LOCALIZATION, AND PHARMACOLOGICAL ROLE OF Kv7 POTASSIUM CHANNELS IN SKELETAL MUSCLE PROLIFERATION, DIFFERENTIATION AND SURVIVAL AFTER MYOTOXIC INSULTS

Changes in the expression of potassium channels regulate skeletal muscle development. The purpose of this study was to investigate the expression profile and pharmacological role of Kv7 voltage-gated potassium channels in skeletal muscle differentiation, proliferation, and survival after myotoxic insults. Transcripts for all Kv7 genes (Kv7.1–Kv7.5) were detected by polymerase chain reaction (PCR) and/or real-time PCR in murine C2C12 myoblasts; Kv7.1, Kv7.3, and Kv7.4 transcripts were up-regulated after myotube formation. Western blot experiments confirmed Kv7.2, Kv7.3, and Kv7.4 subunit expression, and the up-regulation of Kv7.3 and Kv7.4 subunits during in vitro differentiation. In adult skeletal muscles from mice and humans, Kv7.2 and Kv7.3 immunoreactivity was mainly localized at the level of intracellular striations positioned between ankyrinG-positive triads, whereas that of Kv7.4 subunits was largely restricted to the sarcolemmal membrane. In C2C12 cells, retigabine (10 μM), a specific activator of neuronally expressed Kv7.2 to Kv7.5 subunits, reduced proliferation, accelerated myogenin expression, and inhibited the myotoxic effect of mevastatin (IC50 ≈ 7 μM); all these effects of retigabine were prevented by the Kv7 channel blocker 10,10-bis(4-pyridinylmethyl)-9(10H)-anthracenone (XE-991) (10 μM). These data collectively highlight neural Kv7 channels as significant pharmacological targets to regulate skeletal muscle proliferation, differentiation, and myotoxic effects of drugs.