Use of synthetic CaV2.2 Ca2+ channel peptides to study presynaptic function

Small, synthetic peptides based on specific regions of voltage-gated Ca2+ channels (VGCCs) have been widely used to study Ca2+ channel function and have been instrumental in confirming the contribution of specific amino acid sequences to interactions with putative binding partners. In particular, peptides based on the Ca2+ channel Alpha Interaction Domain (AID) on the intracellular region connecting domains I and II (the I-II loop) and the SYNaptic PRotein INTerction (synprint) site on the II-III loop have been widely used. Emerging evidence suggests that such peptides may themselves possess inherent functionality, a property that may be exploitable for future drug design. Here, we review our recent work using synthetic Ca2+ channel peptides based on sequences within the CaV2.2 amino terminal and I-II loop, originally identified as molecular determinates for G protein modulation, and their effects on VGCC function. These CaV2.2 peptides act as inhibitory modules to decrease Ca2+ influx with direct effects on VGCC gating, ultimately leading to a reduction of synaptic transmission. CaV2.2 peptides also attenuate G protein modulation of VGCCs. Amino acid substitutions generate CaV2.2 peptides with increased or decreased inhibitory effects suggesting that synthetic peptides can be used to further probe VGCC function and, potentially, form the basis for novel therapeutic development.

Carbon monoxide inhibition of Cav3.2 T-type Ca2+ channels reveals tonic modulation by thioredoxin

T-type Ca2+ channels play diverse roles in tissues such as sensory neurons, vascular smooth muscle, and cancers, where increased expression of the cytoprotective enzyme, heme oxygenase-1 (HO-1) is often found. Here, we report regulation of T-type Ca2+ channels by carbon monoxide (CO) a HO-1 by-product. CO (applied as CORM-2) caused a concentration-dependent, poorly reversible inhibition of all T-type channel isoforms (Cav3.1-3.3, IC50 ∼3 μM) expressed in HEK293 cells, and native T-type channels in NG108-15 cells and primary rat sensory neurons. No recognized CO-sensitive signaling pathway could account for the CO inhibition of Cav3.2. Instead, CO sensitivity was mediated by an extracellular redox-sensitive site, which was also highly sensitive to thioredoxin (Trx). Trx depletion (using auranofin, 2-5 μM) reduced Cav3.2 currents and their CO sensitivity by >50% but increased sensitivity to dithiothreitol ∼3-fold. By contrast, Cav3.1 and Cav3.3 channels, and their sensitivity to CO, were unaffected in identical experiments. Our data propose a novel signaling pathway in which Trx acts as a tonic, endogenous regulator of Cav3.2 channels, while HO-1-derived CO disrupts this regulation, causing channel inhibition. CO modulation of T-type channels has widespread implications for diverse physiological and pathophysiological mechanisms, such as excitability, contractility, and proliferation

Heme oxygenase-1 regulates cell proliferation via carbon monoxide-mediated inhibition of T-type Ca2+ channels

Induction of the antioxidant enzyme heme oxygenase-1 (HO-1) affords cellular protection and suppresses proliferation of vascular smooth muscle cells (VSMCs) associated with a variety of pathological cardiovascular conditions including myocardial infarction and vascular injury. However, the underlying mechanisms are not fully understood. Over-expression of Cav3.2 T-type Ca2+ channels in HEK293 cells raised basal [Ca2+]i and increased proliferation as compared with non-transfected cells. Proliferation and [Ca2+]i levels were reduced to levels seen in non-transfected cells either by induction of HO-1 or exposure of cells to the HO-1 product, carbon monoxide (CO) (applied as the CO releasing molecule, CORM-3). In the aortic VSMC line A7r5, proliferation was also inhibited by induction of HO-1 or by exposure of cells to CO, and patch-clamp recordings indicated that CO inhibited T-type (as well as L-type) Ca2+ currents in these cells. Finally, in human saphenous vein smooth muscle cells, proliferation was reduced by T-type channel inhibition or by HO-1 induction or CO exposure. The effects of T-type channel blockade and HO-1 induction were non-additive. Collectively, these data indicate that HO-1 regulates proliferation via CO-mediated inhibition of T-type Ca2+ channels. This signalling pathway provides a novel means by which proliferation of VSMCs (and other cells) may be regulated therapeutically.

Hydrogen sulfide inhibits Cav3.2 T-type Ca2+ channels

The importance of H2S as a physiological signaling molecule continues to develop, and ion channels are emerging as a major family of target proteins through which H2S exerts many actions. The purpose of the present study was to investigate its effects on T-type Ca2+ channels. Using patch-clamp electrophysiology, we demonstrate that the H2S donor, NaHS (10 μM-1 mM) selectively inhibits Cav3.2 T-type channels heterologously expressed in HEK293 cells, whereas Cav3.1 and Cav3.3 channels were unaffected. The sensitivity of Cav3.2 channels to H2S required the presence of the redox-sensitive extracellular residue H191, which is also required for tonic binding of Zn2+ to this channel. Chelation of Zn2+ with N,N,N',N'-tetra-2-picolylethylenediamine prevented channel inhibition by H2S and also reversed H2S inhibition when applied after H2S exposure, suggesting that H2S may act via increasing the affinity of the channel for extracellular Zn2+ binding. Inhibition of native T-type channels in 3 cell lines correlated with expression of Cav3.2 and not Cav3.1 channels. Notably, H2S also inhibited native T-type (primarily Cav3.2) channels in sensory dorsal root ganglion neurons. Our data demonstrate a novel target for H2S regulation, the T-type Ca2+ channel Cav3.2, and suggest that such modulation cannot account for the pronociceptive effects of this gasotransmitter.

Identification and expression analyses of Cytosolic Glutamine Synthetase genes in barley (Hordeum vulgare L.)

Glutamine synthetase (GS) is a key enzyme in nitrogen (N) assimilation, particularly during seed development. Three cytosolic GS isoforms (HvGS1) were identified in barley (Hordeum vulgare L. cv Golden Promise). Quantitation of gene expression, localization and response to N supply revealed that each gene plays a non-redundant role in different tissues and during development. Localization of HvGS1_1 in vascular cells of different tissues, combined with its abundance in the stem and its response to changes in N supply, indicate that it is important in N transport and remobilization. HvGS1_1 is located on chromosome 6H at 72.54 cM, close to the marker HVM074 which is associated with a major quantitative trait locus (QTL) for grain protein content (GPC). HvGS1_1 may be a potential candidate gene to manipulate barley GPC. HvGS1_2 mRNA was localized to the leaf mesophyll cells, in the cortex and pericycle of roots, and was the dominant HvGS1 isoform in these tissues. HvGS1_2 expression increased in leaves with an increasing supply of N, suggesting its role in the primary assimilation of N. HvGS1_3 was specifically and predominantly localized in the grain, being highly expressed throughout grain development. HvGS1_3 expression increased specifically in the roots of plants grown on high NH+4, suggesting that it has a primary role in grain N assimilation and also in the protection against ammonium toxicity in roots. The expression of HvGS1 genes is directly correlated with protein and enzymatic activity, indicating that transcriptional regulation is of prime importance in the control of GS activity in barley.