Calcium Currents In Sympathetic Neurons Research Articles

Role of PIP2 in regulating versus modulating Ca2+ channel activity

Role of PIP₂ in regulating <i>versus</i> modulating Ca²⁺ channel activity

Calcium currents and their modulation by strong depolarization in cultured hypertensive and normotensive rat celiac neurons

Calcium channels play an important role in decreased neuron spike accommodation in superior cervical ganglia of spontaneously hypertensive rats. We investigated calcium currents in dissociated celiac neurons from normotensive rats and from rats made hypertensive by treatment with Deoxycorticosterone acetate and high salt diet. Both normotensive and hypertensive celiac neurons showed a high-voltage activated calcium current with peak current at −10mv and reversal potential at +45mv. The currents were totally blocked by 200nM cadmium and 30% blocked by 10μM nifedipine. There were no differences in the amplitude and kinetic properties of these calcium currents between normotensive and hypertensive celiac neurons. G-protein inhibitory modulation of calcium channels is critical in the regulation of calcium currents in rat sympathetic neurons. A brief strong depolarization temporarily reverses G-protein inhibition in superior cervical ganglion. We used a +80mv, 200ms depolarization prior to a 0mv voltage step as the strong depolarization. The strong depolarization did not change the amplitude of calcium currents in neurons from either normotensive or hypertensive rats. However, beginning 2s after the application of the strong depolarization, the peak amplitude of the calcium current was decreased by 20% in neurons from hypertensive rats but was not changed in neurons from normotensive rats. This reduction in current amplitude persisted up to 1 minute. These findings show that strong depolarization induces a long-lasting inhibition of calcium current in sympathetic neurons from hypertensive rats.

Phospholipid metabolism is required for M1 muscarinic inhibition of N-type calcium current in sympathetic neurons.

The signal transduction cascade mediating muscarinic receptor modulation of N-type Ca2+ channel activity by the slow pathway has remained incompletely characterized despite focused investigation. Recently we confirmed a role for the G-protein Gq and identified phospholipase C (PLC), phospholipase A2 (PLA2), and arachidonic acid (AA) as additional molecules involved in N-current inhibition in superior cervical ganglion (SCG) neurons by the slow pathway. We have further characterized this signal transduction cascade by testing whether additional molecules downstream of phosphatidylinositol-4,5-bisphosphate (PIP2) are required. The L-channel antagonist nimodipine was bath-applied to block L-current. Pretreating cells with pertussis toxin (PTX) minimized M2/M4 muscarinic receptor inhibition of N-current by the membrane-delimited pathway. Consistent with our previous studies, pharmacologically antagonizing M1 muscarinic receptors (M1Rs), Gqalpha, PLC, PLA2, and AA minimized N-current inhibition by the muscarinic agonist oxotremorine-M (Oxo-M). When cells were left untreated with PTX, leaving the membrane-delimited pathway intact and the same antagonists retested, Oxo-M decreased whole cell currents. Moreover, inhibited currents displayed slowed activation kinetics, indicating intact N-current inhibition by the membrane-delimited pathway. These findings indicate that the antagonists used to block the slow pathway acted selectively. PLA2 cleaves AA from phospholipids, generating additional metabolites. We tested whether the metabolite lysophosphatidic acid (LPA) mimicked the inhibitory actions of Oxo-M. In contrast to AA, applying LPA did not inhibit whole cell currents. Taken together, these findings suggest that the slow pathway requires M1Rs, Gqalpha, PLC, PIP2, PLA2, and AA for N-current inhibition.

G-proteins and G-protein subunits mediating cholinergic inhibition of N-type calcium currents in sympathetic neurons.

One postsynaptic action of the transmitter acetylcholine in sympathetic ganglia is to inhibit somatic N-type Ca2+ currents: this reduces Ca2+-activated K+ currents and facilitates high-frequency spiking. Previous experiments on rat superior cervical ganglion neurons have revealed two distinct pathways for this inhibitory action: a rapid, voltage-dependent inhibition through activation of M4 muscarinic acetylcholine receptors (mAChRs), and a slower, voltage-independent inhibition via M1 mAChRs [Hille (1994) Trends in Neurosci., 17, 531-536]. We have analysed the mechanistic basis for this divergence at the level of the individual G-proteins and their alpha and betagamma subunits, using a combination of site-directed antibody injection, plasmid-driven antisense RNA expression, overexpression of selected constitutively active subunits, and antagonism of endogenously liberated betagamma subunits by over-expression of Dy-binding P-adrenergic receptor kinase 1 (PARK1) peptide. The results indicate that: (i) M4 mAChR-induced inhibition is mediated by GoA; (ii) a and Py subunits released from the activated GoA heterotrimer produce separate voltage-insensitive and voltage-sensitive components of inhibition, respectively; and (iii) voltage-insensitive M1 mAChR-induced inhibition is likely to be mediated by the alpha subunit of Gq. Hence, Ca2+ current inhibition results from the concerted, but independent actions of three different G-protein subunits.

Closure of potassium M-channels by muscarinic acetylcholine-receptor stimulants requires a diffusible messenger.

The M-current (IK(M)) is a slow voltage-gated K+ current which can be inhibited by muscarinic acetylcholine-receptor (mAChR) agonists. In the present experiments we have tested whether this inhibition results from a local (membrane-delimited) interaction between the receptor and adjacent channels, or whether channel closure is mediated by a diffusible messenger. To do this, single KM(+)-channel currents were recorded from membrane patches in dissociated rat superior cervical sympathetic neurons by using cell-attached patch electrodes. Channel activity was inhibited when muscarine was applied to the cell membrane outside the patch but persisted when channels were exposed to muscarine added to the pipette solution. We conclude that a diffusible molecule (or molecules) is (are) required to induce intrapatch channel closure following activation of extra-patch receptors.