Biochemical and molecular characterisation of the proposed P2Y1-P2Y12 receptor heterodimer in tsA201 cells

The P2Y receptor family represents a wide-ranging set of G-protein-coupled receptors that respond to purinergic ligands, and are involved in many physiological processes. From signalling assays in tsA201 cells, the Kennedy lab has previously proposed the formation of a constitutive heterodimer between co-expressed human P2Y1 and P2Y12 receptors; this could represent a target for the control of pain sensing neurones. Therefore, it was aimed in this project to characterise if the receptors physically interacted using co-immunoprecipitation, and also to investigate the endogenous expression of other potentially interacting hP2Y receptors in the tsA201 cell line. In this study, tsA201 cells were determined to express mRNA for hP2Y1, 2, 4, 6, 12, 14 receptors using reverse transcriptase PCR and confirmatory Sanger sequencing. Furthermore, the cells demonstrated Ca2+ responses to all of the natural hP2Y receptor agonists tested, the order of potency being ADP > ATP > UTP > UDP. When co-expressing hP2Y1 and hP2Y12 receptors oppositely tagged with a HA or fluorescent protein (FP), immunoprecipitating for the HA tag and blotting for the FP showed a visible high molecular weight protein complex of ~130 to >250 KDa for either combination of receptors. This implied that the FP tagged receptor had co-immunoprecipitated with the other HA tagged receptor as this protein was also visible when blotting for the HA tag, signifying the presence of both tagged receptors in the complex. These results were overall suggestive of functional hP2Y1, 2, 12 receptor expression in the tsA201 cell line, and possibly also hP2Y4, 6, 14 receptors. The high molecular weight protein complex detected could represent a constitutive hP2Y1-hP2Y12 receptor heterodimer as proposed. Whilst it was not possible to specifically either confirm or refute the physical formation of the heterodimer with the data acquired, it was potentially supportive, and furthermore suggested other future paths of investigation.

Alternative Splicing in the Voltage-Sensing Region of N-Type CaV2.2 Channels Modulates Channel Kinetics

The CaV2.2 gene encodes the functional core of the N-type calcium channel. This gene has the potential to generate thousands of CaV2.2 splice isoforms with different properties. However, the functional significance of most sites of alternative splicing is not established. The IVS3-IVS4 region contains an alternative splice site that is conserved evolutionarily among CaVα1 genes from Drosophila to human. In CaV2.2, inclusion of exon 31a in the IVS3-IVS4 region is restricted to the peripheral nervous system, and its inclusion slows the speed of channel activation. To investigate the effects of exon 31a in more detail, we generated four tsA201 cell lines stably expressing CaV2.2 splice isoforms. Coexpression of auxiliary CaVβ and CaVα2δ subunits was required to reconstitute currents with the kinetics of N-type channels from neurons. Channels including exon 31a activated and deactivated more slowly at all voltages. Current densities were high enough in the stable cell lines co-expressing CaVα2δ to resolve gating currents. The steady-state voltage dependence of charge movement was not consistently different between splice isoforms, but on gating currents from the exon 31a-containing CaV2.2 isoform decayed with a slower time course, corresponding to slower movement of the charge sensor. Exon 31a-containing CaV2.2 is restricted to peripheral ganglia; and the slower gating kinetics of CaV2.2 splice isoforms containing exon 31a correlated reasonably well with the properties of native N-type currents in sympathetic neurons. Our results suggest that alternative splicing in the S3-S4 linker influences the kinetics but not the voltage dependence of N-type channel gating.

Evidence for a direct interaction between internal tetra-alkylammonium cations and the inactivation gate of cardiac sodium channels.

The effects of internal tetrabutylammonium (TBA) and tetrapentylammonium (TPeA) were studied on human cardiac sodium channels (hH1) expressed in a mammalian tsA201 cell line. Outward currents were measured at positive voltages using a reversed Na gradient. TBA and TPeA cause a concentration-dependent increase in the apparent rate of macroscopic Na current inactivation in response to step depolarizations. At TPeA concentrations < 50 microM the current decay is well fit by a single exponential over a wide voltage range. At higher concentrations a second exponential component is observed, with the fast component being dominant. The blocking and unblocking rate constants of TPeA were estimated from these data, using a three-state kinetic model, and were found to be voltage dependent. The apparent inhibition constant at 0 mV is 9.8 microM, and the blocking site is located 41 +/- 3% of the way into the membrane field from the cytoplasmic side of the channel. Raising the external Na concentration from 10 to 100 mM reduces the TPeA-modified inactivation rates, consistent with a mechanism in which external Na ions displace TPeA from its binding site within the pore. TBA (500 microM) and TPeA (20 microM) induce a use-dependent block of Na channels characterized by a progressive, reversible, decrease in current amplitude in response to trains of depolarizing pulses delivered at 1-s intervals. Tetrapropylammonium (TPrA), a related symmetrical tetra-alkylammonium (TAA), blocks Na currents but does not alter inactivation (O'Leary, M. E., and R. Horn. 1994. Journal of General Physiology. 104:507-522.) or show use dependence. Internal TPrA antagonizes both the TPeA-induced increase in the apparent inactivation rate and the use dependence, suggesting that all TAA compounds share a common binding site in the pore. A channel blocked by TBA or TPeA inactivates at nearly the normal rate, but recovers slowly from inactivation, suggesting that TBA or TPeA in the blocking site can interact directly with a cytoplasmic inactivation gate.