Voltage-dependent Ca2+ channels play a central role in controlling neurotransmitter release at the synapse. They can be inhibited by certain G-protein-coupled receptors, acting by a pathway intrinsic to the membrane. Here we show that this inhibition results from a direct interaction between the G-protein betagamma complex and the pore-forming alpha1 subunits of several types of these channels. The interaction is mediated by the cytoplasmic linker connecting the first and second transmembrane repeats. Within this linker, binding occurs both in the alpha1 interaction domain (AID), which also mediates the interaction between the alpha1 and beta subunits of the channel, and in a second downstream sequence. Further analysis of the binding site showed that several amino-terminal residues in the AID are critical for Gbetagamma binding, defining a site distinct from the carboxy-terminal residues shown to be essential for binding the beta-subunit of the Ca2+ channel. Mutation of an arginine residue within the N-terminal motif abolished betagamma binding and rendered the channel refractory to G-protein modulation when expressed in Xenopus oocytes, showing that the interaction is indeed responsible for G-protein-dependent modulation of Ca2+ channel activity.

The pituitary adenylate cyclase-activating polypeptide (PACAP) receptor is a class II G protein-coupled receptor that contributes to many different cellular functions including neurotransmission, neuronal survival, and synaptic plasticity. The solution structure of the potent antagonist PACAP (residues 6-38) complexed to the N-terminal extracellular (EC) domain of the human splice variant hPAC1-R-short (hPAC1-RS) was determined by NMR. The PACAP peptide adopts a helical conformation when bound to hPAC1-RS with a bend at residue A18 and makes extensive hydrophobic and electrostatic interactions along the exposed ␤-sheet and interconnecting loops of the N-terminal EC domain. Mutagenesis data on both the peptide and the receptor delineate the critical interactions between the C terminus of the peptide and the C terminus of the EC domain that define the high affinity and specificity of hormone binding to hPAC1-RS. These results present a structural basis for hPAC1-RS selectivity for PACAP versus the vasoactive intestinal peptide and also differentiate PACAP residues involved in binding to the N-terminal extracellular domain versus other parts of the full-length hPAC1-RS receptor. The structural, mutational, and binding data are consistent with a model for peptide binding in which the C terminus of the peptide hormone interacts almost exclusively with the N-terminal EC domain, whereas the central region makes contacts to both the N-terminal and other extracellular parts of the receptor, ultimately positioning the N terminus of the peptide to contact the transmembrane region and result in receptor activation.NMR ͉ vasoactive intestinal peptide ͉ G protein-coupled receptor S even transmembrane domain G protein-coupled receptors (GPCRs) are cell surface proteins that transduce signals initiated by hormones or neurotransmitters into the cell (1). Class II GPCRs are a family of receptors that bind structurally related peptide hormones including glucagon, glucagon-like peptides, vasoactive intestinal peptide (VIP), corticotrophinreleasing factor (CRF), parathyroid hormone (PTH), and pituitary adenylate cyclase-activating polypeptide (PACAP) hormone. This family of receptors is capable of regulating intracellular concentrations of cAMP through activation of the adenylate cyclase pathway, and some can also modulate intracellular calcium levels through the phospholipase C pathway. Structurally, they have low homology with other GPCR families but are well conserved within the family. They all contain a relatively large amino-terminal extracellular (EC) domain that plays a critical role in ligand binding. The N-terminal EC hormone-binding domains have common features, typically containing six conserved cysteine residues, two conserved tryptophan residues, and an aspartate residue which has been suggested to be critical for ligand binding (2, 3). Many of the peptide ligands of these receptors have related sequences and can bind to more than one receptor subtype (4).VIP and PACAP are two prototypical neuropeptides that modulate C...

The authentic subunit compositions of neuronal K+ channels purified from bovine brain were analyzed using a monoclonal antibody (mAb 5), reactive exclusively with the Kv1.2 subunit of the latter and polyclonal antibodies specific for fusion proteins containing C-terminal regions of four mammalian Kv proteins. Western blotting of the K+ channels isolated from several brain regions, employing the selective blocker alpha-dendrotoxin (alpha-DTX), revealed the presence in each of four different Kvs. Variable amounts of Kv1.1 and 1.4 subunits were observed in the K+ channels purified from cerebellum, corpus striatum, hippocampus, cerebral cortex, and brain stem; on the other hand, contents of Kv1.6 and 1.2 subunits appeared uniform throughout. Each Kv-specific antibody precipitated a different proportion (anti-Kv1.2 > 1.1 >> 1.6 > 1.4) of the channels detectable with radioiodinated alpha-DTX in every brain region, consistent with a widespread distribution of these oligomeric subtypes. Such heterooligomeric combinations were further documented by the lack of additivity upon their precipitation with a mixture of antibodies to Kv1.1 and Kv1.2; moreover, cross-blotting of the multimers precipitated by mAb 5 showed that they contain all four Kv proteins. Collectively, these findings demonstrate that subtypes of alpha-DTX-susceptible K+ channels are prevalent throughout mammalian brain which are composed of different Kv proteins assembled in complexes, shown previously to also contain auxiliary beta-subunits [Parcej, D. N., Scott, V. E. S., & Dolly, J.O. (1992) Biochemistry 31, 11084-11088].

Neuronal acceptors for alpha-dendrotoxin (alpha-DTX) have recently been purified from mammalian brain and shown to consist of two classes of subunit, a larger (approximately 78,000 M(r)) protein (alpha) whose N-terminal sequence is identical to that of a cloned, alpha-DTX-sensitive K+ channel, and a novel M(r) 39,000 (beta) polypeptide of unknown function. However, little information is available regarding the oligomeric composition of these native molecules. By sedimentation analysis of alpha-DTX acceptors isolated from bovine cortex, two species have been identified. A minority of these oligomers contain only the larger protein, while the vast majority possess both subunits. Based on accurate determination of the molecular weights of these two forms it is proposed that alpha-DTX-sensitive K+ channels exist as alpha 4 beta 4 complexes because this combination gives the best fit to the experimental data.

Voltage-dependent cation channls are large heterooligomeric proteins. Heterologous expression of cDNAs encoding the a subunits alone of K+, Na+, or Ca2+ channels produces functional multimeric proteins; however, coexpression of those for the latter two with their auxiliary proteins causes dramatic changes in the resultant membrane currents. Fast-activating, voltage-sensitive K+ channels from brain contain four a and 13 subunits, tightly associated in a 400-kDa complex; although molecular details of the a-subunit proteins have been determined, little is known about the f-subunit constituent. Proteolytic fragments of a 13 subunit from bovine a-dendrotoxin-sensitive neuronal K+ channels yielded nine different sequences. In the polymerase chain reaction, primers corresponding to two of these peptides amplified a 329-basepair fragment in a AgtlO cDNA library from bovine brain; a full-length clone subsequently isolated encodes a protein of 367 amino acids (Mr 40,983). It shows no significant homology with any known protein. Unlike the channels' a subunits, the hydropathy profile of this sequence failed to reveal transmembrane domains. Several consensus phosphorylation motifs are apparent and, accordingly, the 13 subunit could be phosphorylated in the intact K+ channels. These results, including the absence of a leader sequence and N-glycosylation, are consistent with the 13 subunit being firmly associated on the inside of the membrane with a subunits, as speculated in a simplified model of these authentic K+ channels. Importantly, this first primary structure of a K+-channel 13 subunit indicates that none of the cloned auxiliary proteins of voltage-dependent cation channels, unlike their a subunits, belong to a superfamily of genes.

The ␤ subunit of the voltage-dependent Ca 2؉ channel is a cytoplasmic protein that interacts directly with an ␣ 1 subunit, thereby modulating the biophysical properties of the channel. Herein, we demonstrate that the ␣ 1B subunit of the N-type Ca 2؉ channel associates with several different ␤ subunits. Polyclonal antibodies specific for three different ␤ subunits immunoprecipitated 125 I--conotoxin GVIA binding from solubilized rabbit brain membranes. Enrichment of the N-type Ca 2؉ channels with an ␣ 1B subunit-specific monoclonal antibody showed the association of ␤ 1b , ␤ 3 , and ␤ 4 subunits. Protein sequencing of tryptic peptides of the 57-kDa component of the purified N-type Ca 2؉ channel confirmed the presence of the ␤ 3 and ␤ 4 subunits. Each of the ␤ subunits bound to the ␣ 1B subunit interaction domain with similar high affinity. Thus, our data demonstrate important heterogeneity in the ␤ subunit composition of the N-type Ca 2؉ channels, which may be responsible for some of the diverse kinetic properties recorded from neurons.

TRPA1 is an excitatory, nonselective cation channel implicated in somatosensory function, pain, and neurogenic inflammation. Through covalent modification of cysteine and lysine residues, TRPA1 can be activated by electrophilic compounds, including active ingredients of pungent natural products (e.g., allyl isothiocyanate), environmental irritants (e.g., acrolein), and endogenous ligands (4-hydroxynonenal). However, how covalent modification leads to channel opening is not understood. Here, we report that electrophilic, thioaminal-containing compounds [e.g., CMP1 (4-methyl-N-[2,2,2-trichloro-1-(4-nitro-phenylsulfanyl)-ethyl]-benzamide)] covalently modify cysteine residues but produce striking species-specific effects [i.e., activation of rat TRPA1 (rTRPA1) and blockade of human TRPA1 (hTRPA1) activation by reactive and nonreactive agonists]. Through characterizing rTRPA1 and hTRPA1 chimeric channels and point mutations, we identified several residues in the upper portion of the S6 transmembrane domains as critical determinants of the opposite channel gating: Ala-946 and Met-949 of rTRPA1 determine channel activation, whereas equivalent residues of hTRPA1 (Ser-943 and Ile-946) determine channel block. Furthermore, side-chain replacements at these critical residues profoundly affect channel function. Therefore, our findings reveal a molecular basis of species-specific channel gating and provide novel insights into how TRPA1 respond to stimuli.

N-, P-and Q-type voltage-dependent Ca 2؉ channels control neurotransmitter release in the nervous system and are blocked by -conotoxin MVIIC. In this study, both a high affinity and a low affinity binding site for -conotoxin MVIIC were detected in rabbit brain. The low affinity binding site is shown to be present on the N-type Ca 2؉ channel. Using optimized conditions for specific labeling of the high affinity -conotoxin MVIIC receptor and a panel of subunit specific antibodies, the molecular structure of the high affinity receptor was investigated. We demonstrate for the first time that this receptor is composed of at least ␣ 1A , ␣ 2 ␦, and any one of the four brain ␤ subunits. Such association of different ␤ subunits with ␣ 1A and ␣ 2 ␦ components may produce Ca 2؉ channels with distinct functional properties, such as P-and Q-type. 1 The abbreviations used are: