The heterotrimeric G-protein Gs couples cell-surface receptors to the activation of adenylyl cyclases and cyclic AMP production (reviewed in refs 1, 2). RGS proteins, which act as GTPase-activating proteins (GAPs) for the G-protein alpha-subunits alpha(i) and alpha(q), lack such activity for alpha(s) (refs 3-6). But several RGS proteins inhibit cAMP production by Gs-linked receptors. Here we report that RGS2 reduces cAMP production by odorant-stimulated olfactory epithelium membranes, in which the alpha(s) family member alpha(olf) links odorant receptors to adenylyl cyclase activation. Unexpectedly, RGS2 reduces odorant-elicited cAMP production, not by acting on alpha(olf) but by inhibiting the activity of adenylyl cyclase type III, the predominant adenylyl cyclase isoform in olfactory neurons. Furthermore, whole-cell voltage clamp recordings of odorant-stimulated olfactory neurons indicate that endogenous RGS2 negatively regulates odorant-evoked intracellular signalling. These results reveal a mechanism for controlling the activities of adenylyl cyclases, which probably contributes to the ability of olfactory neurons to discriminate odours.

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 (KV) 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.cGMP ͉ ion channel ͉ signal transduction C yclic nucleotide-gated (CNG) channels sense variations in the intracellular concentration of cyclic nucleotides that occur in response to visual or olfactory stimuli, therefore playing essential roles in the transduction of visual and olfactory information (1, 2). In many ways, CNG channels are similar to voltage-activated potassium (K V ) channels. They coassemble as tetramers of homologous subunits (3-6), each containing six transmembrane segments (TM), a positively charged TM4 and a reentry P region between TM5 and TM6, suggesting that CNG channels belong to the same superfamily of voltage-activated cation channels (7). The main difference is that CNG channels are only weakly voltage-dependent. Instead, they open and close the pore in response to changes in the intracellular concentrations of cGMP or cAMP, a property conferred by the presence of a cyclic nucleotide binding domain at the C terminus of each subunit (8, 9).Our understanding of how CNG channels open and close their pore in response to cyclic nucleotide binding is much less refined than our understanding of how K V channels gate in response to voltage. A large body of evidence, using a variety of approaches, has established that K V channels open and close their permeation pathway at the intracellular end of the pore (10-17). Attempts to extend those ideas to CNG channels have encountered some resistance. For example, studies using intracellularly applied molecules that block the permeation pathway of CNG channels, such as divalent ions (18), tetracaine (19,20), or quaternary ammonium ions (21), have shown that blockade is not state-dependent, as if these molecules can access the pore in both open and closed channels, which is in stark contrast with the blockade properties observed in K V channels (10,11,14,(22)(23)(24). In addition, experiments examining the state dependence of cysteine modification by intracellular application of methanethiosulfonate (MTS) reagents have failed to show dramatic differences between open and closed states in the inner-vestibule region (21,25,26), results that are inconsistent with an intracellular gate in TM6, as shown in K V channels (12,15).Se...

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 (I400 V) alters the inner permeation pathway of human KV1.1, 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.

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.

Editing of Na+/K+ ATPase mRNAs modulates the Na+/K+ pump's turnover rate by selectively targeting the release of the final sodium to the outside.

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.Key Words: RGS, RNA splicing, G-protein, genomic amino-terminal region that shares some similarity with a domain in synapsin [5]. Northern blot analysis revealed prominent 1.8-and 3.5-kb RGS3 mRNA transcripts, but also two lessprominent transcripts of 4.2 and 4.5 kb [5]. The 1.8-and 3.5-kb mRNA transcripts presumably encoded short and long versions of RGS3, but the larger transcripts had no protein-coding region assigned to them. An expression vector for a short form of RGS3 (RGS3T or RGS3CT) was created by arbitrarily choosing an internal ATG [5][6][7]. Both forms of RGS3 act as GAPs for G i␣ and G q␣ , but not G s␣ or G 12␣ [8]. Expression of either form of RGS3 inhibits signaling through a variety of GPCRs that use G i␣ and G q␣ to tranduce signals [5][6][7][8][9]. The shorter version of RGS3 in part localizes in the nucleus and its expression promotes apoptosis of Chinese hamster ovary (CHO) cells [10]. The introduction of RGS3 into B-cell lines inhibited CXCR1-and CXCR4-directed cell migration [11,12]. Perhaps accounting for the efficacy of RGS3 as an inhibitor of chemotaxis, it, in contrast to several other RGS proteins, also inhibits signaling by free G ␤1␥2 subunits [13]. Interference with G ␤␥ signaling blocks chemokine-directed migration [14,15]. 861Article doi:10.1006/geno.2002.6773, available online at http://www.idealibrary.com on IDEAL A partial cDNA for the mouse long form of RGS3 and for a short version of RGS3 with a unique N-terminal 21-amino-acid extension [12] have been identified, as well as a cDNA for the purported full-length version of the long form of mouse RGS3 (GenBank acc. no. AK004648). A partial cDNA clone for another isoform of mouse RGS3, which possesses a 398-amino-acid N-terminal extension containing a PDZ domain, was isolated by interaction cloning using t...

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.

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.