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The symptoms of irritable bowel syndrome (IBS) include significant abdominal pain and bloating. Current treatments are empirical and often poorly efficacious, and there is a need for the development of new and efficient analgesics aimed at IBS patients. T-type calcium channels have previously been validated as a potential target to treat certain neuropathic pain pathologies. Here we report that T-type calcium channels encoded by the Ca V 3.2 isoform are expressed in colonic nociceptive primary afferent neurons and that they contribute to the exaggerated pain perception in a butyratemediated rodent model of IBS. Both the selective genetic inhibition of Ca V 3.2 channels and pharmacological blockade with calcium channel antagonists attenuates IBS-like painful symptoms. Mechanistically, butyrate acts to promote the increased insertion of Ca V 3.2 channels into primary sensory neuron membranes, likely via a posttranslational effect. The butyrate-mediated regulation can be recapitulated with recombinant Ca V 3.2 channels expressed in HEK cells and may provide a convenient in vitro screening system for the identification of T-type channel blockers relevant to visceral pain. These results implicate T-type calcium channels in the pathophysiology of chronic visceral pain and suggest Ca V 3.2 as a promising target for the development of efficient analgesics for the visceral discomfort and pain associated with IBS.analgesia | visceral nociceptor | sensitization | trafficking I rritable bowel syndrome (IBS) is one of the most prevalent lower gastrointestinal (GI) tract disorders, affecting ∼20% of the population in developed countries. Despite high prevalence and considerable impairment of quality of life, current treatments for IBS are empirical and often poorly effective, and the disorder remains a challenge to clinicians (1). IBS is characterized by abdominal pain and discomfort associated with abnormal bowel functions. Although different etiologies have been proposed, it is generally accepted that IBS is multifactorial and that there are likely multiple molecular targets relevant to innovative drug development strategies (2). Among these, there is considerable interest in dysregulation of the brain-gut pain neuraxis and specific subtypes of ion channels in primary afferent neurons that mediate the detection of nociceptive stimuli and transmission to the CNS (3). Moreover, in a number of animal models of chronic pain, the pathological remodeling of ion channel expression patterns has been linked to the hyperexcitability of primary afferent nociceptors (4, 5).A number of ionic conductances contribute to neuronal firing, including voltage-gated calcium channels, which uniquely both shape action potentials and influence neuronal excitability. In mammals, 10 pore-forming calcium channel α 1 subunit genes have been identified, three of which, Ca V 3.1, Ca V 3.2, and Ca V 3.3, form low-voltage-activated (LVA) T-type calcium channels that are activated by weak depolarizations and generally act to control excitability (6, 7). Al...

The disulfide bonds that form between two cysteine residues are important in defining and rigidifying the structures of proteins and peptides. In polypeptides containing multiple cysteine residues, disulfide isomerization can lead to multiple products with different biological activities. Here, we describe the development of a dithiol amino acid (Dtaa) that can form two disulfide bridges at a single amino acid site. Application of Dtaas to a serine protease inhibitor and a nicotinic acetylcholine receptor inhibitor that contain disulfide constraints enhanced their inhibitory activities 40- and 7.6-fold, respectively. X-ray crystallographic and NMR structure analysis show that the peptide ligands containing Dtaas have retained their native tertiary structures. We furthermore show that replacement of two cysteines by Dtaas can avoid the formation of disulfide bond isomers. With these properties, Dtaas are likely to have broad application in the rational design or directed evolution of peptides and proteins with high activity and stability.

Voltage-dependent Ca 2ϩ channels (Ca V ) are membrane proteins that play a key role in promoting Ca 2ϩ influx in response to membrane depolarization in excitable cells. To this date, molecular cloning has identified the primary structures for 10 distinct calcium channel Ca V ␣ 1 subunits (1-7) that are classified into three main subfamilies according to their high voltageactivated (HVA) 2 gating (Ca V 1 and Ca V 2) or low voltage-activated gating (Ca V 3). In addition to the transmembrane poreforming Ca V ␣1 subunit, Ca V 1 and Ca V 2 channels arise from the multimerization of three other proteins (7): a cytoplasmic Ca V ␤ subunit, a mostly extracellular Ca V ␣2␦ subunit, and calmodulin constitutively bound to the C terminus of Ca V ␣1 (8 -12).A considerable body of work documents the interaction and modulation of the Ca V ␣1 subunit of Ca V 1 and Ca V 2 channels (13-18) by the auxiliary Ca V ␤. The high affinity Ca V ␣1-Ca V ␤ interaction site on the pore-forming Ca V ␣1 subunit is a conserved 18-residue sequence in the I-II linker called the ␣ interaction domain (AID) (19,20) that has been structurally resolved by high resolution x-ray crystallography (21-23). Structural work showed that the AID forms a ␣-helix that binds to the ␣ binding pocket (ABP) in the Ca V ␤ nucleotide kinase (NK) domain. It has been proposed that the MMQKAL cluster of residues within the latter determines the high affinity nanomolar interaction between the two proteins (24 -29). Numerous mutational analyses of the AID residues have correlated the Ca V ␤-induced biophysical modulation with the high affinity binding of Ca V ␤ to the AID peptide in a variety of Ca V ␣1 isoforms for Ca V 1 and Ca V 2 channels (25, 29 -32).The association of Ca V ␣1 and Ca V ␤ subunits is also critical for proper channel maturation and cell surface expression of Ca V 2.2 (17), Ca V 1.2 (33, 34), and Ca V 2.3 (35). In Ca V 2.2, the I-II linker is presumed to play a role in this process (17,18), and mutations within the AID motif eliminated its cell surface expression and biophysical modulation by Ca V ␤1b and Ca V ␤3 (32). In addition, the Ca V ␤2-induced increase in Ca V 1.2 whole cell currents was abolished with the AID-defective YWI/AAA mutant (29), suggesting that high affinity binding of Ca V ␤ onto AID is required to traffic Ca V ␣1 to the plasma membrane. Nonetheless, the unique character of the high affinity AID-ABP interface in the membrane targeting of Ca V ␣1 has been questioned (27, 36 -40). In particular, it has been suggested that Ca V ␤-mediated plasma membrane targeting could be uncoupled from Ca V ␤-mediated modulation of channel gating (26, 41) with important contributions from other intracellular regions (33, 39,(42)(43)(44).In addition to Ca V ␤, the ancillary subunit Ca V ␣2␦ and the ubiquitous calmodulin (CaM) protein have also been proposed to modulate HVA channel maturation and targeting (9). For instance, co-expression of Ca V ␣2␦ promoted the trafficking of the Ca V ␣1 subunit of Ca V 2.2 in COS-7 cells (45), suggesting that Ca V ␣2...