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Mechanism of accelerated current decay caused by an episodic ataxia type-1-associated mutant in a potassium channel pore.

ABSTRACT: In Kv1.1, single point mutants found below the channel activation gate at residue V408 are associated with human episodic ataxia type-1, and impair channel function by accelerating decay of outward current during periods of membrane depolarization and channel opening. This decay is usually attributed to C-type inactivation, but here we provide evidence that this is not the case. Using voltage-clamp fluorimetry in Xenopus oocytes, and single-channel patch clamp in mouse ltk- cells, of the homologous Shaker channel (with the equivalent mutation V478A), we have determined that the mutation may cause current decay through a local effect at the activation gate, by destabilizing channel opening. We demonstrate that the effect of the mutant is similar to that of trapped 4-aminopyridine in antagonizing channel opening, as the mutation and 10 mm 4-AP had similar, nonadditive effects on fluorescence recorded from the voltage-sensitive S4 helix. We propose a model where the Kv1.1 activation gate fails to enter a stabilized open conformation, from which the channel would normally C-type inactivate. Instead, the lower pore lining helix is able to enter an activated-not-open conformation during depolarization. These results provide an understanding of the molecular etiology underlying episodic ataxia type-1 due to V408A, as well as biophysical insights into the links between the potassium channel activation gate, the voltage sensor and the selectivity filter.

SUBMITTER: Peters CJ

PROVIDER: S-EPMC6623802 | biostudies-literature | 2011 Nov

REPOSITORIES: biostudies-literature

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Mechanism of accelerated current decay caused by an episodic ataxia type-1-associated mutant in a potassium channel pore.

Peters Christian J CJ Werry Daniel D Gill Hira S HS Accili Eric A EA Fedida David D

The Journal of neuroscience : the official journal of the Society for Neuroscience 20111101 48

In Kv1.1, single point mutants found below the channel activation gate at residue V408 are associated with human episodic ataxia type-1, and impair channel function by accelerating decay of outward current during periods of membrane depolarization and channel opening. This decay is usually attributed to C-type inactivation, but here we provide evidence that this is not the case. Using voltage-clamp fluorimetry in Xenopus oocytes, and single-channel patch clamp in mouse ltk- cells, of the homolog ...[more]

PMID: 22131406

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Project description:SLC1A2 and SLC1A3 encode the glial glutamate transporters EAAT2 and EAAT1, which are not only the predominant glutamate uptake carriers in our brain, but also function as anion channels. Two homologous mutations, which predict substitutions of prolines in the center of the fifth transmembrane helix by arginine (P289R EAAT2, P290R EAAT1), have been identified in patients with epileptic encephalopathy (SLC1A2) or with episodic ataxia type 6 (SLC1A3). Both mutations have been shown to impair glutamate uptake and to increase anion conduction. The molecular processes that link the disease-causing mutations to two major alterations of glutamate transporter function remain insufficiently understood. The mutated proline is conserved in every EAAT. Since the pathogenic changes mainly affect the anion channel function, we here study the functional consequences of the homologous P312R mutation in the neuronal glutamate transporter EAAT4, a low capacity glutamate transporter with predominant anion channel function. To assess the impact of charge and structure of the inserted amino acid for the observed functional changes, we generated and functionally evaluated not only P312R, but also substitutions of P312 with all other amino acids. However, only exchange of proline by arginine, lysine, histidine and asparagine were functionally tolerated. We compared WT, P312R and P312N EAAT4 using a combination of cellular electrophysiology, fast substrate application and kinetic modelling. We found that WT and mutant EAAT4 anion currents can be described with a 11-state model of the transport cycle, in which several states are connected to branching anion channel states to account for the EAAT anion channel function. Substitutions of P312 modify various transitions describing substrate binding/unbinding, translocation or anion channel opening. Most importantly, P312R generates a new anion conducting state that is accessible in the outward facing apo state and that is the main determinant of the increased anion conduction of EAAT transporters carrying this mutation. Our work provides a quantitative description how a naturally occurring mutation changes glutamate uptake and anion currents in two genetic diseases.

Dataset Information

Mechanism of accelerated current decay caused by an episodic ataxia type-1-associated mutant in a potassium channel pore.

Publications

Mechanism of accelerated current decay caused by an episodic ataxia type-1-associated mutant in a potassium channel pore.

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