Ruth Rea

Clinical, genetic, and expression studies of mutations in the potassium channel gene KCNA1 reveal new phenotypic variability.

Episodic ataxia type 1 (EA1) is an autosomal dominant central nervous system potassium channelopathy characterized by brief attacks of cerebellar ataxia and continuous interictal myokymia. Point mutations in the voltage‐gated potassium channel gene KCNA1 on chromosome 12p associate with EA1. We have studied 4 families and identified three new and one previously reported heterozygous point mutations in this gene. Affected members in Family A (KCNA1 G724C) exhibit partial epilepsy and myokymia but no ataxic episodes, supporting the suggestion that there is an association between mutations of KCNA1 and epilepsy. Affected members in Family B (KCNA1 C731A) exhibit myokymia alone, suggesting a new phenotype of isolated myokymia. Family C harbors the first truncation to be reported in KCNA1 (C1249T) and exhibits remarkably drug‐resistant EA1. Affected members in Family D (KCNA1 G1210A) exhibit attacks typical of EA1. This mutation has recently been reported in an apparently unrelated family, although no functional studies were attempted. Heterologous expression of the proteins encoded by the mutant KCNA1 genes suggest that the four point mutations impair delayed‐rectifier type potassium currents by different mechanisms. Increased neuronal excitability is likely to be the common pathophysiological basis for the disease in these families. The degree and nature of the potassium channel dysfunction may be relevant to the new phenotypic observations reported in this study. Ann Neurol 2000;48:647–656

Streamlined synaptic vesicle cycle in cone photoreceptor terminals

Cone photoreceptors tonically release neurotransmitter in the dark through a continuous cycle of exocytosis and endocytosis. Here, using the synaptic vesicle marker FM1-43, we elucidate specialized features of the vesicle cycle. Unlike retinal bipolar cell terminals, where stimulation triggers bulk membrane retrieval, cone terminals appear to exclusively endocytose small vesicles. These retain their integrity until exocytosis, without pooling their membranes in endosomes. Endocytosed vesicles rapidly disperse through the terminal and are reused with no apparent delay. Unlike other synapses where most vesicles are immobilized and held in reserve, only a small fraction (<15%) becomes immobilized in cones. Photobleaching experiments suggest that vesicles move by diffusion and not by molecular motors on the cytoskeleton and that vesicle movement is not rate limiting for release. The huge reservoir of vesicles that move rapidly throughout cone terminals and the lack of a reserve pool are unique features, providing cones with a steady supply for continuous release.

Variable K+ channel subunit dysfunction in inherited mutations of KCNA1

Mutations of KCNA1, which codes for the K+ channel subunit hKv1.1, are associated with the human autosomal dominant disease episodic ataxia type 1 (EA1). Five recently described mutations are associated with a broad range of phenotypes: neuromyotonia alone or with seizures, EA1 with seizures, or very drug‐resistant EA1. Here we investigated the consequences of each mutation for channel assembly, trafficking, gating and permeation. We related data obtained from co‐expression of mutant and wild‐type hKv1.1 to the results of expressing mutant‐wild‐type fusion proteins, and combined electrophysiological recordings in Xenopus oocytes with a pharmacological discrimination of the contribution of mutant and wild‐type subunits to channels expressed at the membrane. We also applied confocal laser scanning microscopy to measure the level of expression of either wild‐type or mutant subunits tagged with green fluorescent protein (GFP). R417stop truncates most of the C‐terminus and is associated with severe drug‐resistant EA1. Electrophysiological and pharmacological measurements indicated that the mutation impairs both tetramerisation of R417stop with wild‐type subunits, and membrane targeting of heterotetramers. This conclusion was supported by confocal laser scanning imaging of enhanced GFP (EGFP)‐tagged hKv1.1 subunits. Co‐expression of R417stop with wild‐type hKv1.2 subunits yielded similar results to co‐expression with wild‐type hKv1.1. Mutations associated with typical EA1 (V404I) or with neuromyotonia alone (P244H) significantly affected neither tetramerisation nor trafficking, and only altered channel kinetics. Two other mutations associated with a severe phenotype (T226R, A242P) yielded an intermediate result. The phenotypic variability of KCNA1 mutations is reflected in a wide range of disorders of channel assembly, trafficking and kinetics.

Encoding Light Intensity by the Cone Photoreceptor Synapse

How cone synapses encode light intensity determines the precision of information transmission at the first synapse on the visual pathway. Although it is known that cone photoreceptors hyperpolarize to light over 4-5 log units of intensity, the relationship between light intensity and transmitter release at the cone synapse has not been determined. Here, we use two-photon microscopy to visualize release of the synaptic vesicle dye FM1-43 from cone terminals in the intact lizard retina, in response to different stimulus light intensities. We then employ electron microscopy to translate these measurements into vesicle release rates. We find that from darkness to bright light, release decreases from 49 to approximately 2 vesicles per 200 ms; therefore, cones compress their 10,000-fold operating range for phototransduction into a 25-fold range for synaptic vesicle release. Tonic release encodes ten distinguishable intensity levels, skewed to most finely represent bright light, assuming release obeys Poisson statistics.

Functional characterization of compound heterozygosity for GlyRα1 mutations in the startle disease hyperekplexia

The human disease hyperekplexia is characterized by excessive startle reactions to auditory and cutaneous stimuli. In its familial form, hyperekplexia has been associated with both dominant and recessive mutations of the GLRA1 gene encoding the glycine receptor α1 subunit (GlyRα1), which mediates inhibitory transmission in the spinal cord and brainstem. Here we have examined the functional consequences of two amino acid substitutions found in a compound heterozygous family, R252H and R392H, to investigate the mechanisms determining this inheritance pattern. When expressed in Xenopus laevis oocytes, both mutations were non‐functional. Neither mutant affected the electrophysiological properties of wild type GlyRα1 when co‐expressed. We introduced a green fluorescent protein tag to mutant subunits and found that both mutant proteins were detectable. Evidence that subcellular localization differed from wild type was significant for one of the mutants. Thus, an effective loss of functional GlyRα1‐mediated current underlies hyperekplexia in this family, whereas a partial loss is asymptomatic.

The Inherited Episodic Ataxias: How Well Do We Understand the Disease Mechanisms?

The past few years have seen the elucidation of several neurological diseases caused by inherited mutations of ion channels. In contrast to many other types of genetic disorders, the “channelopathies” can be studied with high precision by applying electrophysiological methods. This review evaluates the success of this approach in explaining the mechanisms of two forms of episodic ataxia that are known to be caused by mutations of ion channels: episodic ataxia type 1 (EA1, caused by K+ channel mutations) and episodic ataxia type 2 (EA2, caused by Ca2+ channel mutations). Although both of these disorders are rare, they raise many important questions about the roles of identified channels in brain function. Indeed, a resolution of the mechanisms by which both diseases occur will represent a major milestone in understanding diseases of the CNS, in addition to opening the way to novel possible treatments.