Mutations in the KCNT1 (Slack, K Na 1.1) sodium-activated potassium channel produce severe epileptic encephalopathies. Expression in heterologous systems has shown that the disease-causing mutations give rise to channels that have increased current amplitude. It is not known, however, whether such gain of function occurs in human neurons, nor whether such increased K Na current is expected to suppress or increase the excitability of cortical neurons. Using genetically engineered human induced pluripotent stem cell (iPSC)-derived neurons, we have now found that sodium-dependent potassium currents are increased several-fold in neurons bearing a homozygous P924L mutation. In current-clamp recordings, the increased K Na current in neurons with the P924L mutation acts to shorten the duration of action potentials and to increase the amplitude of the afterhyperpolarization that follows each action potential. Strikingly, the number of action potentials that were evoked by depolarizing currents as well as maximal firing rates were increased in neurons expressing the mutant channel. In networks of spontaneously active neurons, the mean firing rate, the occurrence of rapid bursts of action potentials, and the intensity of firing during the burst were all increased in neurons with the P924L Slack mutation. The feasibility of an increased K Na current to increase firing rates independent of any compensatory changes was validated by numerical simulations. Our findings indicate that gain-of-function in Slack K Na channels causes hyperexcitability in both isolated neurons and in neural networks and occurs by a cell-autonomous mechanism that does not require network interactions.

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Heterozygous NRXN1 deletions constitute the most prevalent currently known single-gene mutation associated with schizophrenia, and additionally predispose to multiple other neurodevelopmental disorders. Engineered heterozygous NRXN1 deletions impaired neurotransmitter release in human neurons, suggesting a synaptic pathophysiological mechanism. Utilizing this observation for drug discovery, however, requires confidence in its robustness and validity. Here, we describe a multicenter effort to test the generality of this pivotal observation, using independent analyses at two laboratories of patient-derived and newly engineered human neurons with heterozygous NRXN1 deletions. Using neurons transdifferentiated from induced pluripotent stem cells that were derived from schizophrenia patients carrying heterozygous NRXN1 deletions, we observed the same synaptic impairment as in engineered NRXN1-deficient neurons. This impairment manifested as a large decrease in spontaneous synaptic events, in evoked synaptic responses, and in synaptic paired-pulse depression. Nrxn1-deficient mouse neurons generated from embryonic stem cells by the same method as human neurons did not exhibit impaired neurotransmitter release, suggesting a human-specific phenotype. Human NRXN1 deletions produced a reproducible increase in the levels of CASK, an intracellular NRXN1-binding protein, and were associated with characteristic gene-expression changes. Thus, heterozygous NRXN1 deletions robustly impair synaptic function in human neurons regardless of genetic background, enabling future drug discovery efforts.

Phosphite dehydrogenase represents a new enzymatic system for regenerating reduced nicotinamide cofactors for industrial biocatalysis. We previously engineered a variant of phosphite dehydrogenase with relaxed cofactor specificity and significantly increased activity and stability. Here we performed one round of random mutagenesis followed by comprehensive saturation mutagenesis to further improve the enzyme thermostability while maintaining its activity. Two new thermostabilizing mutations were identified. These, along with the 12 mutations previously identified, were subjected to saturation mutagenesis using the parent enzyme or the engineered thermostable variant 12x as a template, followed by screening of variants with increased thermostability. Of the 12 previously identified sites, 6 yielded new variants with improved stability over the parent enzyme. Several mutations were found to be context-dependent. On the basis of molecular modeling and biochemical analysis, various mechanisms of thermostabilization were identified. Combining the most thermostabilizing mutation at each site resulted in a variant that showed a 100-fold increase in half-life at 62 degrees C over the 12x mutant. The final mutant has improved the half-life of thermal inactivation at 45 degrees C by 23,000-fold over the parent enzyme. The engineered phosphite dehydrogenase will be useful in NAD(P)H regeneration.