Regulation of Na+/K+ ATPase transport velocity by RNA editing.

Because firing properties and metabolic rates vary widely, neurons require different transport rates from their Na(+)/K(+) pumps in order to maintain ion homeostasis. In this study we show that Na(+)/K(+) pump activity is tightly regulated by a novel process, RNA editing. Three codons within the squid Na(+)/K(+) ATPase gene can be recoded at the RNA level, and the efficiency of conversion for each varies dramatically, and independently, between tissues. At one site, a highly conserved isoleucine in the seventh transmembrane span can be converted to a valine, a change that shifts the pump's intrinsic voltage dependence. Mechanistically, the removal of a single methyl group specifically targets the process of Na(+) release to the extracellular solution, causing a higher turnover rate at the resting membrane potential.

Structural basis of Na(+)/K(+)-ATPase adaptation to marine environments.

Throughout evolution, enzymes have adapted to perform in different environments. The Na(+)/K(+) pump, an enzyme crucial for maintaining ionic gradients across cell membranes, is strongly influenced by the ionic environment. In vertebrates, the pump sees much less external Na(+) (100-160 mM) than it does in osmoconformers such as squid (450 mM), which live in seawater. If the extracellular architecture of the squid pump were identical to that of vertebrates, then at the resting potential, the pump's function would be severely compromised because the negative voltage would drive Na(+) ions back to their binding sites, practically abolishing forward transport. Here we show that four amino acids that ring the external mouth of the ion translocation pathway are more positive in squid, thereby reducing the pump's sensitivity to external Na(+) and explaining how it can perform optimally in the marine environment.

Gating at the selectivity filter in cyclic nucleotide-gated channels.

By opening and closing the permeation pathway (gating) in response to cGMP binding, cyclic nucleotide-gated (CNG) channels serve key roles in the transduction of visual and olfactory signals. Compiling evidence suggests that the activation gate in CNG channels is not located at the intracellular end of pore, as it has been established for voltage-activated potassium (K(V)) channels. Here, we show that ion permeation in CNG channels is tightly regulated at the selectivity filter. By scanning the entire selectivity filter using small cysteine reagents, like cadmium and silver, we observed a state-dependent accessibility pattern consistent with gated access at the middle of the selectivity filter, likely at the corresponding position known to regulate structural changes in KcsA channels in response to low concentrations of permeant ions.

Demonstration of ion channel synthesis by isolated squid giant axon provides functional evidence for localized axonal membrane protein translation.

Local translation of membrane proteins in neuronal subcellular domains like soma, dendrites and axon termini is well-documented. In this study, we isolated the electrical signaling unit of an axon by dissecting giant axons from mature squids (Dosidicus gigas). Axoplasm extracted from these axons was found to contain ribosomal RNAs, ~8000 messenger RNA species, many encoding the translation machinery, membrane proteins, translocon and signal recognition particle (SRP) subunits, endomembrane-associated proteins, and unprecedented proportions of SRP RNA (~68% identical to human homolog). While these components support endoplasmic reticulum-dependent protein synthesis, functional assessment of a newly synthesized membrane protein in axolemma of an isolated axon is technically challenging. Ion channels are ideal proteins for this purpose because their functional dynamics can be directly evaluated by applying voltage clamp across the axon membrane. We delivered in vitro transcribed RNA encoding native or Drosophila voltage-activated Shaker KV channel into excised squid giant axons. We found that total K+ currents increased in both cases; with added inactivation kinetics on those axons injected with RNA encoding the Shaker channel. These results provide unambiguous evidence that isolated axons can exhibit de novo synthesis, assembly and membrane incorporation of fully functional oligomeric membrane proteins.

Quasi-specific access of the potassium channel inactivation gate.

Many voltage-gated potassium channels open in response to membrane depolarization and then inactivate within milliseconds. Neurons use these channels to tune their excitability. In Shaker K(+) channels, inactivation is caused by the cytoplasmic amino terminus, termed the inactivation gate. Despite having four such gates, inactivation is caused by the movement of a single gate into a position that occludes ion permeation. The pathway that this single inactivation gate takes into its inactivating position remains unknown. Here we show that a single gate threads through the intracellular entryway of its own subunit, but the tip of the gate has sufficient freedom to interact with all four subunits deep in the pore, and does so with equal probability. This pathway demonstrates that flexibility afforded by the inactivation peptide segment at the tip of the N-terminus is used to mediate function.

Editing of human K(V)1.1 channel mRNAs disrupts binding of the N-terminus tip at the intracellular cavity.

In the nervous system, A→I RNA editing has an important role in regulating neuronal excitability. Ligand-gated membrane receptors, synaptic proteins, as well as ion channels, are targets for recoding by RNA editing. Although scores of editing sites have been identified in the mammalian brain, little is known about the functional alterations that they cause, and even less about the mechanistic underpinnings of how they change protein function. We have previously shown that an RNA editing event (I,400 V) alters the inner permeation pathway of human K(V)1.1, modifying the kinetics of fast inactivation. Here we show that the channel's inactivation gate enters deep into the ion permeation pathway and the very tip establishes a direct hydrophobic interaction with the edited position. By converting I to V, the intimacy of the interaction is reduced, allowing the inactivation gate to unbind with much faster kinetics.

Additional 5' exons in the RGS3 locus generate multiple mRNA transcripts, one of which accounts for the origin of human PDZ-RGS3.

Regulators of G-protein signaling (RGS) proteins can be broadly divided into those that consist predominantly of an RGS domain and those that possess an RGS domain along with additional domains. RGS3 fits into both categories, as both short and longer forms exist. Recently, a novel form of mouse RGS3 that possesses a PDZ domain was identified. Here we show that the human PDZ-RGS3 isoform arises from 10 upstream exons along with 6 exons from the previously characterized RGS3. We found that 47,000 nucleotides span the last of the 10 upstream exons and the first exon used from the original cluster of RGS3 exons. These 10 upstream exons encode 398 amino acids, which show strong conservation with those from mouse PDZ-RGS3. In addition, another isoform exists that uses 17 upstream exons, 9 of which overlap with those in PDZ-RGS3, along with the same 6 downstream exons used in PDZ-RGS3. Finally, a short form of human RGS3 arises from an unrecognized RGS3 exon that encodes an amino-terminal 140 amino acids. For each RGS3 isoform, RT-PCR detected specific mRNA transcripts and immunoblot analysis identified specific bands for RGS3 and PDZ-RGS3. RGS3 provides an example of the complex origins of the coding regions of mammalian proteins.