Cysteine string proteins (CSPs) are secretory vesicle proteins bearing a "J domain" and a palmitoylated cysteine-rich "string" region that are critical for neurotransmitter release. The precise role of CSP in neurotransmission is controversial. Here, we demonstrate a novel interaction between CSP, receptor-coupled trimeric GTP binding proteins (G proteins), and N-type Ca2+ channels. G. subunits interact with the J domain of CSP in an ATP-dependent manner; in contrast, Gbetagamma subunits interact with the C terminus of CSP in both the presence and absence of ATP. The interaction of CSP with both G proteins and N-type Ca2+ channels results in a tonic G protein inhibition of the channels. In view of the crucial importance of N-type Ca2+ channels in presynaptic vesicle release, our data attribute a key role to CSP in the fine tuning of neurotransmission.

Using transient calcium phosphate transfection into the human embryonic kidney tsa‐201 cell line and subsequent whole‐cell patch‐clamp protocols, we examined the tonic modulation of cloned N‐ and P/Q‐type calcium channels by five different G protein β subunits via strong depolarizing voltage prepulses. For N‐ and P/Q‐type channels, the magnitude of inhibition was dependent on the Gβ subtype co‐expressed. Both the absolute and relative magnitudes of Gβ subunit‐induced inhibition of P/Q‐type channels differed from those observed with the N‐type channel. For each calcium channel subtype, kinetics of both the prepulse‐mediated recovery from inhibition and the re‐inhibition following the prepulse were examined for each of the Gβ subunits by varying either the duration between the pre‐ and the test pulse or the length of the prepulse. For each channel subtype, we observed a differential Gβ subunit rank order with regard to the rates of re‐inhibition and recovery from inhibition. On average, P/Q‐type channels exhibited more rapid rates of recovery from inhibition than those observed with N‐type channels. Different Gβ subtypes mediated different degrees of slowing of activation kinetics. The differential modulation of P/Q‐ and N‐type channels by various Gβ subtypes may provide a mechanism for fine tuning the amount of calcium entering the presynaptic nerve termini.

The direct inhibition of N-and P/Q-type calcium channels by G protein ␤␥ subunits is considered a key mechanism for regulating presynaptic calcium levels. We have recently reported that a number of features associated with this G protein inhibition are dependent on the G protein ␤ subunit isoform (Arnot, M. I., Stotz, S. C., Jarvis, S. E., Zamponi, G. W. Chem. 275, 40777-40781). Here, we have examined the abilities of different types of ancillary calcium channel ␤ subunits to modulate the inhibition of ␣ 1B N-type calcium channels by the five known different G␤ subunit subtypes. Our data reveal that the degree of inhibition by a particular G␤ subunit is strongly dependent on the specific calcium channel ␤ subunit, with N-type channels containing the ␤ 4 subunit being less susceptible to G␤␥-induced inhibition. The calcium channel ␤ 2a subunit uniquely slows the kinetics of recovery from G protein inhibition, in addition to mediating a dramatic enhancement of the G protein-induced kinetic slowing. For G␤ 3 -mediated inhibition, the latter effect is reduced following site-directed mutagenesis of two palmitoylation sites in the ␤ 2a N-terminal region, suggesting that the unique membrane tethering of this subunit serves to modulate G protein inhibition of N-type calcium channels. Taken together, our data suggest that the nature of the calcium channel ␤ subunit present is an important determinant of G protein inhibition of N-type channels, thereby providing a possible mechanism by which the cellular/subcellular expression pattern of the four calcium channel ␤ subunits may regulate the G protein sensitivity of N-type channels expressed at different loci throughout the brain and possibly within a neuron.The inhibition of presynaptic calcium channels by the activation of seven-helix transmembrane receptors is an important mechanism for modulating calcium influx into presynaptic nerve termini. It is now well established that this inhibitory mechanism occurs via a direct interaction between the G protein ␤␥ dimer and the calcium channel ␣ 1 subunit (Refs. 1 and 2; for review, see Ref.3). Upon G␤␥ binding, the channels experience an increase in first latency to opening (4), which is seen as a decrease in peak current amplitude, a slowing of the time course of activation, and a slowed macroscopic time course of inactivation at the whole cell level. The inhibition can be relieved by application of a strong depolarizing voltage prepulse (5), which causes a transient dissociation of the G protein complex from the channel (6) and, consequently, a temporary restoration of normal current kinetics. The overall degree of inhibition is strongly dependent not only on the nature of the calcium channel ␣ 1 subunit (7-10), but also on the G␤ subunit subtype (10 -12). The notion of a G␤ subunit isoform dependence of this G protein effect is also supported by the observation that protein kinase C is able to antagonize the inhibition of N-type calcium channels by G␤ 1 subunits, but not that mediated by other G␤ subunit isoforms (13).It is ...

The modulation of N-type calcium current by protein kinases and G-proteins is a factor in the fine tuning of neurotransmitter release. We have previously shown that phosphorylation of threonine 422 in the ␣ 1B calcium channel domain I-II linker region resulted in a dramatic reduction in somatostatin receptor-mediated G-protein inhibition of the channels and that the I-II linker consequently serves as an integration center for cross-talk between protein kinase C (PKC) and G-proteins (Hamid, J., Nelson, D., Spaetgens, R., Dubel, S. J., Snutch, T. P., and Zamponi, G. W. (1999) J. Biol. Chem. 274, 6195-6202). Here we show that opioid receptor-mediated inhibition of N-type channels is affected to a lesser extent compared with that seen with somatostatin receptors, hinting at the possibility that PKC/G-protein cross-talk might be dependent on the G-protein subtype. To address this issue, we have examined the effects of four different types of G-protein ␤ subunits on both wild type and mutant ␣ 1B calcium channels in which residue 422 has been replaced by glutamate to mimic PKC-dependent phosphorylation and on channels that have been directly phosphorylated by protein kinase C. Our data show that phosphorylation or mutation of residue 422 antagonizes the effect of G␤ 1 on channel activity, whereas G␤ 2 , G␤ 3 , and G␤ 4 are not affected. Our data therefore suggest that the observed cross-talk between G-proteins and protein kinase C modulation of N-type channels is a selective feature of the G␤ 1 subunit.The modulation of calcium channel activity by activation of intracellular messenger pathways is a key mechanism for fine tuning calcium entry into neurons. For example, the activation of protein kinase C has been shown to mediate an up-regulation of N-type calcium currents in intact neurons (1, 2) and in transient expression systems (3,4). In contrast, the direct 1:1 binding of G protein ␤␥ subunits to the domain I-II linker region of N-type, P/Q-type, and possibly R-type calcium channels results in a depression of current activity (5-8) (reviewed in Refs. 9 and 10), which can be reversed by strong membrane depolarization (10 -12). Different types of calcium channels are modulated by G-proteins to different extents, such that N-type channels are typically inhibited more effectively than P/Q-type channels (13-16). There is also increasing evidence that the degree of inhibition is dependent on the G-protein ␤ subunit species (16 -18). Finally, it has been shown that protein kinase C-dependent phosphorylation of the N-type calcium channel ␣ 1 subunit antagonizes receptor-mediated G-protein inhibition of the channel (1,2,12,19). This phenomenon (termed PKC 1 /Gprotein cross-talk) appears to be mediated by a single threonine residue in the ␣ 1B domain I-II linker region (4). For somatostatin receptor-induced G-protein inhibition of N-type calcium channels, mutation of Thr-422 to glutamic acid mimics the antagonistic effect of protein kinase C on G protein inhibition, whereas a switch to alanine precludes the occurrence of P...

It has been asserted that, when screening chemicals for bioaccumulation potential, molecular size cutoff criteria (or indicators) can be applied above which no, or limited, bioaccumulation is expected. The suggested molecular size values have increased over time as more measurements have become available. Most of the proposed criteria have been derived from unevaluated fish bioconcentration factor (BCF) data, and less than 5% of existing organic substances have measured BCFs. We critically review the proposed criteria, first by considering other factors that may also contribute to reduced bioaccumulation for larger molecules, namely, reduced bioavailability in the water column, reduced rate of uptake corresponding to reduced diffusion rates, and the effects of biotransformation and growth dilution. An evaluated BCF and bioaccumulation factor (BAF) database for more than 700 substances and dietary uptake efficiency data are compared against proposed cutoff values. We examine errors associated with interpreting BCF data, particularly for developing molecular size criteria of bioaccumulation potential. Reduced bioaccumulation that is often associated with larger molecular size can be explained by factors other than molecular size, and there is evidence of absorption of molecules exceeding the proposed cutoff criteria. The available data do not support strict cutoff criteria, indicating that the proposed values are incorrect. Rather than assessing bioaccumulation using specific chemical properties in isolation, holistic methods that account for competing rates of uptake and elimination in an organism are recommended. An integrated testing strategy is suggested to improve knowledge of the absorption and bioaccumulation of large substances. Integr Environ Assess Manag 2010;6:210-224. ß 2009 SETAC