T-type calcium channels are essential contributors to the transmission of nociceptive signals in the primary afferent pain pathway. Here, we show that T-type calcium channels are ubiquitinated by WWP1, a plasma-membrane-associated ubiquitin ligase that binds to the intracellular domain III-IV linker region of the Cav3.2 T-type channel and modifies specific lysine residues in this region. A proteomic screen identified the deubiquitinating enzyme USP5 as a Cav3.2 III-IV linker interacting partner. Knockdown of USP5 via shRNA increases Cav3.2 ubiquitination, decreases Cav3.2 protein levels, and reduces Cav3.2 whole-cell currents. In vivo knockdown of USP5 or uncoupling USP5 from native Cav3.2 channels via intrathecal delivery of Tat peptides mediates analgesia in both inflammatory and neuropathic mouse models of mechanical hypersensitivity. Altogether, our experiments reveal a cell signaling pathway that regulates T-type channel activity and their role in nociceptive signaling.

Objective:The relationship between genetic variation in the T-type calcium channel gene CACNA1H and childhood absence epilepsy is well established. The purpose of this study was to investigate the range of epilepsy syndromes for which CACNA1H variants may contribute to the genetic susceptibility architecture and determine the electrophysiological effects of these variants in relation to proposed mechanisms underlying seizures. Methods: Exons 3 to 35 of CACNA1H were screened for variants in 240 epilepsy patients (167 unrelated) and 95 control subjects by single-stranded conformation analysis followed by direct sequencing. Cascade testing of families was done by sequencing or single-stranded conformation analysis. Selected variants were introduced into the CACNA1H protein by sitedirected mutagenesis. Constructs were transiently transfected into human embryo kidney cells, and electrophysiological data were acquired.

Potassium channels that are inhibited by internal ATP (KATP channels) provide a critical link between metabolism and cellular excitability. Protein kinase C (PKC) acts on K ATP channels to regulate diverse cellular processes, including cardioprotection by ischemic preconditioning and pancreatic insulin secretion. PKC action decreases the Hill coefficient of ATP binding to cardiac K ATP channels, thereby increasing their open probability at physiological ATP concentrations. We show that PKC similarly regulates recombinant channels from both the pancreas and heart. Surprisingly, PKC acts via phosphorylation of a specific, conserved threonine residue (T180) in the pore-forming subunit (Kir6.2). Additional PKC consensus sites exist on both Kir and the larger sulfonylurea receptor (SUR) subunits. Nonetheless, T180 controls changes in open probability induced by direct PKC action either in the absence of, or in complex with, the accessory SUR1 (pancreatic) or SUR2A (cardiac) subunits. The high degree of conservation of this site among different K ATP channel isoforms suggests that this pathway may have wide significance for the physiological regulation of KATP channels in various tissues and organelles. P otassium channels that are inhibited by ATP (K ATP channels) consist of a heterooctamer of four sulfonylurea receptor (SURx) and four inwardly rectifying K ϩ channel (Kir6.x) subunits (1-5). The SUR is a member of the ATP-binding cassette (ABC) family of proteins and acts as a regulatory subunit, conferring ADP sensitivity and the distinctive pharmacological characteristics on the K ATP channel complex (1-6). In contrast, the Kir6.x subunit forms the pore of the channel and mediates the defining ATP-dependent inhibition of K ATP channels (6). Protein kinase-catalyzed phosphorylation is an important mechanism by which the activity of ion channels, including the K ATP channel, can be controlled (7-9). For instance, another ABC protein ion channel, the cystic fibrosis transmembrane conductance regulator, is regulated by cAMP-dependent protein kinase-mediated phosphorylation, which, itself, may be permissively regulated by protein kinase C (PKC) (8, 10, 11). In addition, mounting evidence suggests the importance of PKC in activating K ATP channels during both the protective mechanism of ischemic preconditioning (12, 13) and in regulating insulin secretion (14), although the site(s) and mechanism of action of PKC-mediated phosphorylation events have not been described. Therefore, we sought to determine: (i) the functional effects of PKC on the K ATP channel, (ii) whether the action of PKC is mediated via the SUR or Kir6.2 subunit, and (iii) the identity of specific amino acid residue(s) phosphorylated by PKC. Materials and MethodsCell Culture and Transfection. tsA201 cells (an SV40-transformed variant of the HEK293 human embryonic kidney cell line) were maintained in DMEM supplemented with 10 mM glucose͞2 mM L-glutamine͞10% FCS͞0.1% penicillin/streptomycin at 37°C (10% CO 2 ). Cells were plated at 30-40% confluence on 35-...

We examined ion channels derived from a chloroform extract of isolated, dehydrated rat liver mitochondria. The extraction method was previously used to isolate a channel-forming complex containing poly-3-hydroxybutyrate and calcium polyphosphate from Escherichia coli. This complex is also present in eukaryotic membranes, and is located primarily in mitochondria. Reconstituted channels showed multiple subconductance levels and were voltage-dependent, showing an increased probability of higher conductance states at voltages near zero. In symmetric 150 mM KCl, the maximal conductance of the channel ranged from 350 pS to 750 pS. For voltages >+/-60 mV, conductance fluctuated in the range of approximately 50- approximately 200 pS. In the presence of a 1:3 gradient of KCl, at pH = 7.4, selectivity periodically switched between different states ranging from weakly anion-selective (V(rev) approximately -15 mV) to ideally cation-selective (V(rev) approximately +29 mV), without a significant change in its conductance. Overall, the diverse, but highly reproducible, channel activity most closely resembled the behavior of the permeability transition pore channel seen in patch-clamp experiments on native mitoplasts. We suggest that the isolated complex may represent the ion-conducting module from the permeability transition pore.

Kinetics and voltage dependence of inactivation of a prokaryotic voltage-gated sodium channel (NaChBac) were investigated in an effort to understand its molecular mechanism. NaChBac inactivation kinetics show strong, bell-shaped voltage dependence with characteristic time constants ranging from approximately 50 ms at depolarized voltages to a maximum of approximately 100 s at the inactivation midpoint. Activation and inactivation parameters for four different covalently linked tandem dimer or tandem tetramer constructs were indistinguishable from those of the wild-type channel. Point mutations in the outer part of the pore revealed an important influence of the S195 residue on the process of inactivation. For two mutants (S195D and S195E), the maximal and minimal rates of inactivation observed were increased by approximately 2.5-fold, and the midpoint of the steady-state inactivation curve was shifted approximately 20 mV in the hyperpolarizing direction, compared to the wild-type channel. Our data suggest that pore vestibule structure is an important determinant of NaChBac inactivation, whereas the inactivation mechanism is independent of the number of free cytoplasmic N- and C-termini in the functional channel. In these respects, NaChBac inactivation resembles C-type or slow inactivation modes observed in other voltage-gated K and Na channels.