Insights into channel function via channel dysfunction

The nicotinic synapse has been a touchstone for advances in neuroscience ever since Jean Nicot, the French ambassador to Portugal, sent some tobacco seeds home to Paris in 1550 with a note that the New World plant had interesting effects when smoked. Now the muscle nicotinic acetylcholine receptor (nAChR) is a well-studied example of ligand-gated ion channels. After a motor neuron is stimulated, the nerve impulse reaches the presynaptic terminal, where it evokes release of acetylcholine (ACh) into the synapse. The nAChR depolarizes the postsynaptic muscle and triggers muscle action potentials; muscle contraction follows. To date, several nAChR subtypes have been successfully isolated, purified, imaged, and expressed, and unitary currents have been recorded from these channels (1). Researchers continue to unravel the molecular mechanisms of these macromolecules that are embedded in membranes at vertebrate nerve-muscle synapses, at invertebrate nicotinic synapses (which explains why nicotine-producing tobacco plants have a select advantage against invertebrate pests), and in the vertebrate central system (which explains Jean Nicot’s fascination with those leaves). However, the precise structural events that trigger channel opening or "gating" remain mostly unknown

Mutations Linked to Autosomal Dominant Nocturnal Frontal Lobe Epilepsy Affect Allosteric Ca²⁺ Activation of the α4β2 Nicotinic Acetylcholine Receptor

Extracellular Ca²⁺ robustly potentiates the acetylcholine response of α4β2 nicotinic receptors. Rat orthologs of five mutations linked to autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE)—α4(S252F), α4(S256L), α4(+L264), β2(V262L), and β2(V262M)—reduced 2 mM Ca²⁺ potentiation of the α4β2 1 mM acetylcholine response by 55 to 74%. To determine whether altered allosteric Ca²⁺ activation or enhanced Ca²⁺ block caused this reduction, we coexpressed the rat ADNFLE mutations with an α4 N-terminal mutation, α4(E180Q), that abolished α4β2 allosteric Ca²⁺ activation. In each case, Ca²⁺ inhibition of the double mutants was less than that expected from a Ca²⁺ blocking mechanism. In fact, the effects of Ca²⁺ on the ADNFLE mutations near the intracellular end of the M2 region—α4(S252F) and α4(S256L)—were consistent with a straightforward allosteric mechanism. In contrast, the effects of Ca²⁺ on the ADNFLE mutations near the extracellular end of the M2 region—α4(+L264)β2, β2(V262L), and β2(V262M)—were consistent with a mixed mechanism involving both altered allosteric activation and enhanced block. However, the effects of 2 mM Ca²⁺ on the α4β2, α4(+L264)β2, and α4β2(V262L) single-channel conductances, the effects of membrane potential on the β2(V262L)-mediated reduction in Ca²⁺ potentiation, and the effects of eliminating the negative charges in the extracellular ring on this reduction failed to provide any direct evidence of mutant-enhanced Ca²⁺ block. Moreover, analyses of the α4β2, α4(S256L), and α4(+L264) Ca²⁺ concentration-potentiation relations suggested that the ADNFLE mutations reduce Ca²⁺ potentiation of the α4β2 acetylcholine response by altering allosteric activation rather than by enhancing block

Novel Seizure Phenotype and Sleep Disruptions in Knock-In Mice with Hypersensitive α4* Nicotinic Receptors

A leucine to alanine substitution (L9′A) was introduced in the M2 region of the mouse α4 neuronal nicotinic acetylcholine receptor (nAChR) subunit. Expressed in Xenopus oocytes, α4(L9′A)β2 nAChRs were ≥30-fold more sensitive than wild type (WT) to both ACh and nicotine. We generated knock-in mice with the L9′A mutation and studied their cellular responses, seizure phenotype, and sleep-wake cycle. Seizure studies on α4-mutated animals are relevant to epilepsy research because all known mutations linked to autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE) occur in the M2 region of α4or β2 subunits. Thalamic cultures and synaptosomes from L9′A mice were hypersensitive to nicotine-induced ion flux. L9′A mice were ∼15-fold more sensitive to seizures elicited by nicotine injection than their WT littermates. Seizures in L9′A mice differed qualitatively from those in WT: L9′A seizures started earlier, were prevented by nicotine pretreatment, lacked EEG spike-wave discharges, and consisted of fast repetitive movements. Nicotine-induced seizures in L9′A mice were partial, whereas WT seizures were generalized. When L9′A homozygous mice received a 10 mg/kg nicotine injection, there was temporal and phenomenological separation of mutant and WT-like seizures: an initial seizure ∼20 s after injection was clonic and showed no EEG changes. A second seizure began 3-4 min after injection, was tonic-clonic, and had EEG spike-wave activity. No spontaneous seizures were detected in L9′A mice during chronic video/EEG recordings, but their sleep-wake cycle was altered. Our findings show that hypersensitive α4* nicotinic receptors in mice mediate changes in the sleep-wake cycle and nicotine-induced seizures resembling ADNFLE

Five ADNFLE Mutations Reduce the Ca²⁺ Dependence of the Mammalian α4β2 Acetylcholine Response

Five nicotinic acetylcholine receptor (nAChR) mutations are currently linked to autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE). The similarity of their clinical symptoms suggests that a common functional anomaly of the mutations underlies ADNFLE seizures. To identify this anomaly, we constructed rat orthologues (S252F, +L264, S256L, V262L, V262M) of the human ADNFLE mutations, expressed them in Xenopus oocytes with the appropriate wild‐type (WT) subunit (α4 or β2), and studied the Ca²⁺ dependence of their ACh responses. All the mutations significantly reduced 2 mM Ca²⁺‐induced increases in the 30 μM ACh response (P < 0.05). Consistent with a dominant mode of inheritance, this reduction persisted in oocytes injected with a 1:1 mixture of mutant and WT cRNA. BAPTA injections showed that the reduction was not due to a decrease in the secondary activation of Ca²⁺‐activated Cl⁻ currents. The S256L mutation also abolished 2 mM Ba²⁺ potentiation of the ACh response. The S256L, V262L and V262M mutations had complex effects on the ACh concentration‐response relationship but all three mutations shifted the concentration‐response relationship to the left at [ACh]⩾ 30 μM. Co‐expression of the V262M mutation with a mutation (E180Q) that abolished Ca²⁺ potentiation resulted in 2 mM Ca²⁺ block, rather than potentiation, of the 30 μM ACh response, suggesting that the ADNFLE mutations reduce Ca²⁺ potentiation by enhancing Ca²⁺ block of the α4β2 nAChR. Ca²⁺ modulation may prevent presynaptic α4β2 nAChRs from overstimulating glutamate release at central excitatory synapses during bouts of synchronous, repetitive activity. Reducing the Ca²⁺ dependence of the ACh response could trigger seizures by increasing α4β2‐mediated glutamate release during such bouts

Five ADNFLE mutations reduce the Ca2+ dependence of the mammalian α4β2 acetylcholine response

Five nicotinic acetylcholine receptor (nAChR) mutations are currently linked to autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE). The similarity of their clinical symptoms suggests that a common functional anomaly of the mutations underlies ADNFLE seizures. To identify this anomaly, we constructed rat orthologues (S252F, +L264, S256L, V262L, V262M) of the human ADNFLE mutations, expressed them in Xenopus oocytes with the appropriate wild-type (WT) subunit (α4 or β2), and studied the Ca2+ dependence of their ACh responses. All the mutations significantly reduced 2 mM Ca2+-induced increases in the 30 μM ACh response (P < 0.05). Consistent with a dominant mode of inheritance, this reduction persisted in oocytes injected with a 1:1 mixture of mutant and WT cRNA. BAPTA injections showed that the reduction was not due to a decrease in the secondary activation of Ca2+-activated Cl− currents. The S256L mutation also abolished 2 mM Ba2+ potentiation of the ACh response. The S256L, V262L and V262M mutations had complex effects on the ACh concentration-response relationship but all three mutations shifted the concentration-response relationship to the left at [ACh]≥ 30 μM. Co-expression of the V262M mutation with a mutation (E180Q) that abolished Ca2+ potentiation resulted in 2 mM Ca2+ block, rather than potentiation, of the 30 μM ACh response, suggesting that the ADNFLE mutations reduce Ca2+ potentiation by enhancing Ca2+ block of the α4β2 nAChR. Ca2+ modulation may prevent presynaptic α4β2 nAChRs from overstimulating glutamate release at central excitatory synapses during bouts of synchronous, repetitive activity. Reducing the Ca2+ dependence of the ACh response could trigger seizures by increasing α4β2-mediated glutamate release during such bouts