Structure and Function of Membrane Proteins

National Institute Of Neurological Disorders And Stroke

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Abstract

Voltage-activated potassium (KV) channels are potassium selective integral membrane proteins formed by the assembly of four homologous subunits. Large-conductance voltage- and calcium- dependent potassium (BK) channels is a KV channel in which the open probability is regulated by the transmembrane voltage and the concentration of intracellular Ca2+. Voltage is sensed by the voltage sensor embedded in the cell membrane while Ca2+ binds to sites located at a large cytoplasmic C-terminus region called the gating ring. We use simultaneous optical and electrophysiological recordings to study the interplay between dynamic changes of the gating ring in response to Ca2+ binding and the conformational changes associated with channel activation. To understand, at a molecular and biophysical level, the conformational changes of the gating ring in response to Ca2+ binding, and how these movements are coupled to the opening and closing of the gate of the channel, we combine cysteine substitution following chemical modification and/or disulfide bond formation with the patch clamp fluorometry technique; which allows us to simultaneously monitor ion currents through the pore and FRET signals derived from fluorescent proteins inserted within the gating ring. Using published cryo-EM structures of the entire BK channels we target regions in which the transmembrane segments S2-S5 appear to be interacting with the gating ring. We are also beginning to implement a preparation to perform experiments with BK channels at the single molecule level, using fluorescence approaches. There is an unparalleled understanding of a system once you study it at the single molecule level. Our goal is to perform single molecule Fluorescence Resonance Energy Transfer (smFRET). We also study the gating mechanisms of transporters, like the Na/K-ATPase. This enzyme, a member of the P-type family (named for their phosphorylated intermediates), harnesses the energy from the hydrolysis of one ATP to alternately export 3Na ions and import 2K ions against their electrochemical gradients. By performing this active transport, the Na/K pump plays an essential role in the homeostasis of intracellular Na and K that is crucial to sustaining cell excitability, volume, and Na-dependent secondary transport. On the basis of biochemical data accumulated during the decade following its discovery, the Na/K-ATPase was proposed to alternately transport Na and K ions according to a model known as the Post-Albers scheme. As ions are transported through the Na/K pump, they become temporarily occluded within the protein, inaccessible from either side, before being released. By restricting Na/K pumps to only the reversible transitions associated with deocclusion and extracellular release of Na+, it is possible to detect pre-steady state electrical signals accompanying those transitions. The signals arise because Na+ traverse a fraction of the membrane potential as they enter or leave their binding sites deep within the pump. At a fixed membrane potential and external sodium concentration, the populations of pumps with empty binding sites, and those with bound or occluded Na, reach a steady-state distribution. A sudden change of membrane voltage then shifts the Na-binding equilibrium, and initiates a redistribution of the pump populations towards a new steady state. The consequent change in Na-binding-site occupancy causes Na to travel between the extracellular environment and the pump interior. In so doing they generate a current. As the system approaches a new steady distribution, fewer Na move, and the current declines. The electrical signals therefore appear as transient currents. Using the squid giant axon preparation, which exploits axial current delivery to generate very fast membrane voltage steps, we previously identified three phases of relaxation in transient pump currents (Holmgren et al., 2000; Gadsby et al., 2012): fast (comparable to the voltage-jump time course), medium-speed (tm 0.2-0.5 ms), and slow (ts 1-10 ms). We suggested that each phase reflects a distinct Na-binding event (or release, depending on the direction of the voltage change) with its associated conformational transition (occlusion or deocclusion). In other words, the Na/K-ATPase undergoes dynamic rearrangements that open external gates to allow bound Na access to the extracellular environment immediately prior to release. This work with squid giant axons has set the foundations for our present study attempting to understand the functional consequences of mutations within human Na/K-ATPase isoforms. Recently, genetic mutations in the brain specific Na/K-ATPase (ATP1A3) have been linked to specific human pathologies, like Alternating Hemiplegia of Childhood (AHC), a devastating disease affecting over 120 unrelated children around the world. We have begun to study the functional consequences of some of these mutations, in particular D801N, E815K and G947R. These positions are located within the transmembrane region of the ATP1A3. We hypothesize that because of their location, they might influence ion binding transitions. In addition, we have acquired a knock-in mice for the most common ATP1A3 mutation causing AHC (D801N), which we intend to use to perform behavioral experiments. Unfortunately, given the COVID-19 restrictions, we have only been able to monitor their survival. The ion channel activity of the ORF3a protein from previous SARS-CoVs has been related to the activation of the inflammasome and apoptosis of infected cells, suggesting a relevance of this protein in the pathogenesis of COVID-19. Frequent non-synonymous mutations are detected in the ORF3a from SARS-CoV-2 with a possible gain or loss of channel function, which might have a novel influence on the virus pathogenicity. We are studying the properties of ORF3a by three different approaches: 1) synthesis and trafficking using imaging, 2) biophysical properties of ion channel activity using electrophysiology, and 3) stoichiometry using single molecule fluorescence. Once the properties of this specific ORF3a protein have been characterized, we will standardize them for comparison with functional properties from other mutants of ORF3a proteins observed in others SARS-CoV-2 strains.