Oscillatory firing patterns are an intrinsic property of some neurons and have an important function in information processing. In some cells, low voltage-activated calcium channels have been proposed to underlie a depolarizing potential that regulates bursting. The sequence of a rat brain calcium channel alpha 1 subunit (rbE-II) was deduced. Although it is structurally related to high voltage-activated calcium channels, the rbE-II channel transiently activated at negative membrane potentials, required a strong hyperpolarization to deinactivate, and was highly sensitive to block by nickel. In situ hybridization showed that rbE-II messenger RNA is expressed in regions throughout the central nervous system. The electrophysiological properties of the rbE-II current are consistent with a type of low voltage-activated calcium channel that requires membrane hyperpolarization for maximal activity, which suggests that rbE-II may be involved in the modulation of firing patterns.

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In vitro display technologies, best exemplified by phage and yeast display, were first described for the selection of antibodies some twenty years ago. Since that time a large number of antibodies, some with remarkable properties, have been selected and improved upon using these methods. The first antibodies derived using in vitro display methods are now in the clinic, with many more waiting in the wings. Here we discuss the scope of the technology, some of the powerful antibodies selected, and the future potential in a post-genomic world. Unique advantages offered by in vitro display technologies include the ability to carefully define selection conditions, allowing the derivation of antibodies recognizing predefined epitopes or conformations, the further improvement of selected antibodies, the potential for high throughput applications and the immediate availability of genes encoding the selected antibody. We anticipate that the high throughput potential of these technologies will soon lead to their use to select antibodies against all human proteins.

Functional expression of the rat brain alA Ca channel was obtained by nuclear injection of an expression plasmid into Xenopus oocytes. The alA Ca current activated quickly, inactivated slowly, and showed a voltage dependence typical of high voltage-activated Ca channels. The alA current was partially blocked (=23%) by w-agatoxin IVA (200 nM) and substantially blocked by a-conotoxin MVIIC (5 pM blocked "70%). Bay K 8644 (10 pM) or a-conotoxin GVIA (1 IM) had no significant effect on the alA current. Coexpression with rat brain Ca channel 13 subunits increased the alA whole-cell current and shifted the current-voltage relation to more negative values. While the f18b and (3 subunits caused a significant acceleration of the alA inactivation kinetics, the 82. subunit dramatically slowed the inactivation of the alA current to that seen typically for P-type Ca currents. In situ loaliztion with antisense deoxyoligonucleotide and RNA probes showed that alA was widely distributed throughout the rat central nervous system, with moderate to high levels in the olfactory bulb, in the cerebral cortex, and in the CA fields and dentate gyrus of the hippocampus. In the cerebellum, prominent alA expression was detected in Purkuije cells with some labeling also in granule cells. Overall, the results show that aj channels are widely expressed and share some properties with both Qand P-type channels.

The T-type calcium channel Cav3.2 emerges as a key regulator of sensory functions, but its expression pattern within primary afferent neurons and its contribution to modality-specific signaling remain obscure. Here, we elucidate this issue using a unique knockin/flox mouse strain wherein Cav3.2 is replaced by a functional Cav3.2-surface-ecliptic GFP fusion. We demonstrate that Cav3.2 is a selective marker of two major low-threshold mechanoreceptors (LTMRs), Aδ- and C-LTMRs, innervating the most abundant skin hair follicles. The presence of Cav3.2 along LTMR-fiber trajectories is consistent with critical roles at multiple sites, setting their strong excitability. Strikingly, the C-LTMR-specific knockout uncovers that Cav3.2 regulates light-touch perception and noxious mechanical cold and chemical sensations and is essential to build up that debilitates allodynic symptoms of neuropathic pain, a mechanism thought to be entirely A-LTMR specific. Collectively, our findings support a fundamental role for Cav3.2 in touch/pain pathophysiology, validating their critic pharmacological relevance to relieve mechanical and cold allodynia.