Francesco Miceli

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.

Coupling between the voltage-sensing and phosphatase domains of Ci-VSP

The Ciona intestinalis voltage sensor–containing phosphatase (Ci-VSP) shares high homology with the phosphatidylinositol phosphatase enzyme known as PTEN (phosphatase and tensin homologue deleted on chromosome 10). We have taken advantage of the similarity between these proteins to inquire about the coupling between the voltage sensing and the phosphatase domains in Ci-VSP. Recently, it was shown that four basic residues (R11, K13, R14, and R15) in PTEN are critical for its binding onto the membrane, required for its catalytic activity. Ci-VSP has three of the basic residues of PTEN. Here, we show that when R253 and R254 (which are the homologues of R14 and R15 in PTEN) are mutated to alanines in Ci-VSP, phosphatase activity is disrupted, as revealed by a lack of effect on the ionic currents of KCNQ2/3, where current decrease is a measure of phosphatase activity. The enzymatic activity was not rescued by the introduction of lysines, indicating that the binding is an arginine-specific interaction between the phosphatase binding domain and the membrane, presumably through the phosphate groups of the phospholipids. We also found that the kinetics and steady-state voltage dependence of the S4 segment movement are affected when the arginines are not present, indicating that the interaction of R253 and R254 with the membrane, required for the catalytic action of the phosphatase, restricts the movement of the voltage sensor.

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.

Involvement of KCNQ2 subunits in [3H]dopamine release triggered by depolarization and pre-synaptic muscarinic receptor activation from rat striatal synaptosomes

KCNQ2 and KCNQ3 subunits encode for the muscarinic‐regulated current (IKM), a sub‐threshold voltage‐dependent K+ current regulating neuronal excitability. In this study, we have investigated the involvement of IKM in dopamine (DA) release from rat striatal synaptosomes evoked by elevated extracellular K+ concentrations ([K+]e) and by muscarinic receptor activation. [3H]dopamine ([3H]DA) release triggered by 9 mmol/L [K+]e was inhibited by the IKM activator retigabine (0.01–30 μmol/L; Emax = 54.80 ± 3.85%; IC50 = 0.50 ± 0.36 μmol/L). The IKM blockers tetraethylammonium (0.1–3 mmol/L) and XE‐991 (0.1–30 μmol/L) enhanced K+‐evoked [3H]DA release and prevented retigabine‐induced inhibition of depolarization‐evoked [3H]DA release. Retigabine‐induced inhibition of K+‐evoked [3H]DA release was also abolished by synaptosomal entrapment of blocking anti‐KCNQ2 polyclonal antibodies, an effect prevented by antibody pre‐absorption with the KCNQ2 immunizing peptide. Furthermore, the cholinergic agonist oxotremorine (OXO) (1–300 μmol/L) potentiated 9 mmol/L [K+]e‐evoked [3H]DA release (Emax = 155 ± 9.50%; EC50 = 25 ± 1.80 μmol/L). OXO (100 μmol/L)‐induced [3H]DA release enhancement was competitively inhibited by pirenzepine (1–10 nmol/L) and abolished by the M3‐preferring antagonist 4‐diphenylacetoxy N‐methylpiperidine methiodide (1 μmol/L), but was unaffected by the M1‐selective antagonist MT‐7 (10–100 nmol/L) or by Pertussis toxin (1.5–3 μg/mL), which uncouples M2‐ and M4‐mediated responses. Finally, OXO‐induced potentiation of depolarization‐induced [3H]DA release was not additive to that produced by XE‐991 (10 μmol/L), was unaffected by retigabine (10 μmol/L), and was abolished by synaptosomal entrapment of anti‐KCNQ2 antibodies. Collectively, these findings indicate that, in rat striatal nerve endings, IKM channels containing KCNQ2 subunits regulate depolarization‐induced DA release and that IKM suppression is involved in the reinforcement of depolarization‐induced DA release triggered by the activation of pre‐synaptic muscarinic heteroreceptors.

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.

IKs channels open slowly because KCNE1 accessory subunits slow the movement of S4 voltage sensors in KCNQ1 pore-forming subunits

Significance E1 and Q1 protein subunits assemble to form IKslow channels in the heart and ear. Inherited mutations in either subunit that decrease protein level or alter function can cause life-threatening cardiac arrhythmias and deafness. The mechanism by which E1 slows channel opening has been the subject of active debate. Here, we use gating current measurements and simultaneous recordings of ionic currents and changes in fluorescence of a probe on the Q1 voltage sensors to demonstrate that E1 slows the movement of sensors in a manner that is both necessary and sufficient to determine the slow activation time course of IKs channels. Human IKs channels activate slowly with the onset of cardiac action potentials to repolarize the myocardium. IKs channels are composed of KCNQ1 (Q1) pore-forming subunits that carry S4 voltage-sensor segments and KCNE1 (E1) accessory subunits. Together, Q1 and E1 subunits recapitulate the conductive and kinetic properties of IKs. How E1 modulates Q1 has been unclear. Investigators have variously posited that E1 slows the movement of S4 segments, slows opening and closing of the conduction pore, or modifies both aspects of electromechanical coupling. Here, we show that Q1 gating current can be resolved in the absence of E1, but not in its presence, consistent with slowed movement of the voltage sensor. E1 was directly demonstrated to slow S4 movement with a fluorescent probe on the Q1 voltage sensor. Direct correlation of the kinetics of S4 motion and ionic current indicated that slowing of sensor movement by E1 was both necessary and sufficient to determine the slow-activation time course of IKs.

Correlating the Clinical and Genetic Features of Benign Familial Neonatal Seizures (BFNS) with the Functional Consequences of Underlying Mutations

Almost ten years have passed since the identification of Kv7.2 and Kv7.3, the genes altered in Benign Familial Neonatal Seizures (BFNS), a familial autosomal dominant focal epilepsy of the newborn. Despite the rarity of the disease, clinical and genetic data have been gathered from more than 50 BFNS-affected families; these studies reveal that each family harbours a specific disease-causing mutation, and that the mutation-induced functional changes range from a subtle alteration in channel behaviour to a complete ablation of channel function. Prompted by the recent identification of peculiar gating changes in Kv7.2 subunits caused by novel mutations responsible for BFNS, in the present work we attempt to link, whenever possible, the specific genetic defect with the clinical evolution of the disease in the affected families on one side, and, on the other, with the functional defects revealed by expression studies. Such genotype-phenotype correlations may provide clues on the pathogenesis of the wide variety of neuropsychiatric manifestations often associated to BFNS, and should foster our attempts to gain more detailed functional information which might help to elucidate the pathogenetic mechanisms of the disease.