Stephen R. Ikeda

Double-pulse calcium channel current facilitation in adult rat sympathetic neurones

1. Double‐pulse facilitation of Ca2+ channel currents in enzymatically dispersed adult rat superior cervical ganglion neurones was investigated using the whole‐cell variant of the patch‐clamp technique. Voltage‐clamp recordings were performed at room temperature (21‐24 degrees C) in solutions designed to isolate Ca2+ channel currents. 2. Ba2+ currents, elicited by a 0 mV test pulse, were increased in amplitude when preceded by a 40 ms pulse to voltages greater than 0 mV. The magnitude of facilitation was dependent on pre‐pulse voltage and reached a maximum of 50% (i.e. 1.5 x the current amplitude elicited without a pre‐pulse) at a pre‐pulse voltage of +80 mV. Half‐maximal facilitation occurred at about +25 mV. A small decrease (‐6%) in test pulse amplitude was present at pre‐pulse voltages between ‐40 and 0 mV. The magnitude of facilitation was also dependent on test pulse voltage. Facilitation was greatest between test pulse voltages of ‐10 and 0 mV. 3. Facilitation slowly decreased during prolonged (1 h) dialysis of the neurone even though the Ba2+ current amplitude was well maintained. 4. Increasing the pre‐pulse duration over the range 0‐120 ms produced an exponential increase in facilitation with a time constant of 17.3 ms. Conversely, lengthening the interpulse duration over the range 5‐915 ms, while maintaining a constant pre‐pulse amplitude and duration, resulted in an exponential decrease in facilitation with a time constant of 197 ms. 5. At a test potential of 0 mV, the decay of the facilitated Ba2+ current component was fitted to a double exponential function with time constants of about 25 and 150 ms. The time constants had little pre‐pulse voltage dependence between +30 to +80 mV. 6. The initial rising phase of both the control and facilitated Ba2+ current were reasonably well described by a single exponential (tau rise) after a delay of 300 microseconds. The tau rise versus test pulse potential relationship was ‘bell shaped’ over the test pulse voltage of ‐20 to +30 mV reaching a maximum near ‐5 mV. tau rise was similar for control and facilitated currents except at potentials greater than +10 mV where the rise of the facilitated current was accelerated. 7. Control and facilitated activation curves, as derived from tail current amplitudes, were described by the sum of two Boltzmann functions. A facilitating pre‐pulse produced an increase in the proportion of the current contributed by the component activated at more hyperpolarized test potentials.(ABSTRACT TRUNCATED AT 400 WORDS)

Homer proteins regulate coupling of group I metabotropic glutamate receptors to N-type calcium and M-type potassium channels.

Group I metabotropic glutamate receptors (mGluR1 and 5) couple to intracellular calcium pools by a family of proteins, termed Homer, that cross-link the receptor to inositol trisphosphate receptors. mGluRs also couple to membrane ion channels via G-proteins. The role of Homer proteins in channel modulation was investigated by expressing mGluRs and various forms of Homer in rat superior cervical ganglion (SCG) sympathetic neurons by intranuclear cDNA injection. Expression of cross-linking-capable forms of Homer (Homer 1b, 1c, 2, and 3, termed long forms) occluded group I mGluR-mediated N-type calcium and M-type potassium current modulation. This effect was specific for group I mGluRs. mGluR2 (group II)-mediated inhibition of N-channels was unaltered. Long forms of Homer decreased modulation of N- and M-type currents but did not selectively block distinct G-protein pathways. Short forms of Homer, which cannot self-multimerize (Homer 1a and a Homer 2 C-terminal deletion), did not alter mGluR–ion channel coupling. When coexpressed with long forms of Homer, short forms restored the mGluR1a-mediated calcium current modulation in an apparent dose-dependent manner. Homer 2b induced cell surface clusters of mGluR5 in SCG neurons. Conversely, a uniform distribution was observed when mGluR5 was expressed alone or with Homer short forms. These studies indicate that long and short forms of Homer compete for binding to mGluRs and regulate their coupling to ion channels. In vivo, the immediate early Homer 1a is anticipated to enhance ion channel modulation and to disrupt coupling to releasable intracellular calcium pools. Thus, Homer may regulate the magnitude and predominate signaling output of group I mGluRs.

Heterologous expression of metabotropic glutamate receptors in adult rat sympathetic neurons: Subtype-specific coupling to ion channels

A novel heterologous expression system was used to examine the coupling of metabotropic glutamate receptors (mGluRs) to neuronal voltage-gated ion channels. Cytoplasmic injection of mGluR2 cRNA into adult rat sympathetic neurons resulted in the expression of receptors that negatively coupled to N-type Ca2+ channels through a pertussis toxin-sensitive pathway. Injection of mGluR1 alpha cRNA resulted in the expression of receptors that inhibited M-type K+ channels via a pertussis toxin-insensitive pathway. Coupling was restricted to specific transduction elements and effectors, since mGluR2 did not inhibit M channels and mGluR1 alpha had minimal effects on Ca2+ channels. These findings demonstrate that heterologously expressed, and thus unambiguously identified, mGluR subtypes modulate specific neuronal ion channels through discrete signal transduction pathways.

Expression of RGS2 Alters the Coupling of Metabotropic Glutamate Receptor 1a to M-Type K+ and N-Type Ca2+ Channels

Group I mGluRs heterologously expressed in sympathetic neurons inhibited calcium (I(Ca)) and M-type potassium (I(M)) currents. Treatment with pertussis toxin (PTX) revealed a voltage-dependent (VD), PTX-sensitive component of I(Ca) inhibition and a voltage-independent (VI), PTX-insensitive component. Coexpression of RGS2 occluded mGluR1a inhibition of I(M) and made I(Ca) inhibition VD in PTX-treated cells, presumably by blocking the effects of G alpha(q/11)-GTP. These data indicate that mGluR1a can couple to G(i/o) as well as G(q/11). In addition, VI I(Ca) inhibition proceeds through a G alpha(q/11)-GTP-mediated pathway, which can be occluded by expressing RGS2, leaving the VD, G betagamma-mediated inhibition active. These data may reveal a functional role for the upregulation of RGS2 expression in in vivo systems.

G Protein α Subunit Gαz Couples Neurotransmitter Receptors to Ion Channels in Sympathetic Neurons

Abstract The functional roles subserved by Gα z , a G protein α subunit found predominantly in neuronal tissues, have remained largely undefined. Here, we report that Gα z coupled neurotransmitter receptors to N-type Ca 2+ channels when transiently overexpressed in rat sympathetic neurons. The Gα z -mediated inhibition was voltage dependent and PTX insensitive. Recovery from Gα z -mediated inhibition was extremely slow but accelerated by coexpression with RGS proteins. Gα z selectively interacted with a subset of receptors that ordinarily couple to N-type Ca 2+ channels via PTX-sensitive G o/i proteins. In addition, Gα z rescued the activation of heterologously expressed GIRK channels in PTX-treated neurons. These results suggest that Gα z is capable of coupling receptors to ion channels and might underlie PTX-insensitive ion channel modulation observed in neurons under physiological and pathological conditions.

VIP inhibits N-type Ca2+ channels of sympathetic neurons via a pertussis toxin-insensitive but cholera toxin-sensitive pathway

The best characterized Ca2+ channel modulation in mammalian sympathetic neurons is an inhibition of N-type channels via a pertussis toxin (PTX)-sensitive heterotrimeric G protein. Here, we show that vasoactive intestinal polypeptide (VIP), an abundant neuropeptide in the PNS and CNS, inhibited N-type Ca2+ channels in rat sympathetic neurons in a voltage-dependent, membrane-delimited manner. The effect of VIP was insensitive to PTX but was attenuated by cholera toxin or anti-Gs alpha antibodies. VIP-mediated inhibition was independent of cAMP-dependent protein kinase A (PKA). The results provide evidence for a new signal transduction pathway in which N-type Ca2+ channel modulation requires activation of Gs alpha but is independent of PKA-mediated phosphorylation.

Tetrodotoxin‐resistant sodium current of rat nodose neurones: monovalent cation selectivity and divalent cation block.

1. Monovalent cation selectivity and divalent cation sensitivity of the tetrodotoxin (TTX)‐resistant Na+ current in dissociated adult rat nodose ganglion neurones were investigated using the whole‐cell patch‐clamp technique. 2. The TTX‐resistant Na+ current was isolated using ion substitution and pharmacological agents. Under these conditions, the current reversal potential shifted 52 mV per tenfold change in external [Na+]. 3. Inorganic and organic monovalent cation permeability ratios (Px/PNa) were determined from changes in reversal potential and the Goldman‐Hodgkin‐Katz equation. The Px/PNa values determined by the former method were HONH3+, 1.38; Li+, 1.00; H2NNH3+, 0.66; NH4+, 0.28; CH3NH3+, less than 0.13; K+, less than 0.13; Rb+, less than 0.12; Cs+, less than 0.10; (CH3)4N+, less than 0.10. The values determined by either method agreed within 10%. 4. The effects of Cd2+, Co2+, Mn2+ and Ni2+ on the TTX‐resistant Na+ current were analysed from peak‐conductance values. These ions shifted the activation of the current to more positive potentials and decreased the maximal conductance. At 3 mM concentrations, Cd2+, Ni2+, Co2+ and Mn2+ decreased the maximal conductance 64.6, 50.7, 25.0 and 20.3%, respectively. 5. The results indicate that: (a) the monovalent cation selectivity of the TTX‐resistant Na+ current is similar to that of the TTX‐sensitive Na+ current in other tissues; and (b) the TTX‐resistant Na+ current is less sensitive to divalent cations than the Ca2+ current in these neurones. These observations suggest that the structure determining the monovalent cation permeability of the TTX‐resistant Na+ current is similar to that of the TTX‐sensitive Na+ current in other tissues, and that the channels carrying the TTX‐resistant Na+ current are distinct from those responsible for the Ca2+ current.

Expression of Rem2, an RGK family small GTPase, reduces N-type calcium current without affecting channel surface density.

Rad, Gem/Kir, Rem, and Rem2 are members of the Ras-related RGK (Rad, Gem, and Kir) family of small GTP-binding proteins. Heterologous expression of RGK proteins interferes with de novo calcium channel assembly/trafficking and dramatically decreases the amplitude of currents arising from preexisting high-voltage-activated calcium channels. These effects probably result from the direct interaction of RGK proteins with calcium channel β subunits. Among the RGK family, Rem2 is the only member abundantly expressed in neuronal tissues. Here, we examined the ability of Rem2 to modulate endogenous voltage-activated calcium channels in rat sympathetic and dorsal root ganglion neurons. Heterologous expression of Rem2 nearly abolished calcium currents arising from preexisting high-voltage-activated calcium channels without affecting low-voltage-activated calcium channels. Rem2 inhibition of N-type calcium channels required both the Ras homology (core) domain and the polybasic C terminus. Mutation of a putative GTP/Mg2+ binding motif in Rem2 did not affect suppression of calcium currents. Loading neurons with GDP-β-S via the patch pipette did not reverse Rem2-mediated calcium channel inhibition. Finally, [125I]Tyr22-ω-conotoxin GVIA cell surface binding in tsA201 cells stably expressing N-type calcium channels was not altered by Rem2 expression at a time when calcium current was totally abolished. Together, our results support a model in which Rem2 localizes to the plasma membrane via a C-terminal polybasic motif and interacts with calcium channel β subunits in the preassembled N-type channel, thereby forming a nonconducting species.

Phosphorylation of Critical Serine Residues in Gem Separates Cytoskeletal Reorganization from Down-Regulation of Calcium Channel Activity

ABSTRACT Gem is a small GTP-binding protein that has a ras-like core and extended chains at each terminus. The primary structure of Gem and other RGK family members (Rad, Rem, and Rem2) predicts a GTPase deficiency, leading to the question of how Gem functional activity is regulated. Two functions for Gem have been demonstrated, including inhibition of voltage-gated calcium channel activity and inhibition of Rho kinase-mediated cytoskeletal reorganization, such as stress fiber formation and neurite retraction. These functions for Gem have been ascribed to its interaction with the calcium channel β subunit and Rho kinase β, respectively. We show here that these functions are separable and regulated by distinct structural modifications to Gem. Phosphorylation of serines 261 and 289, located in the C-terminal extension, is required for Gem-mediated cytoskeletal reorganization, while GTP and possibly calmodulin binding are required for calcium channel inhibition. In addition to regulating cytoskeletal reorganization, phosphorylation of serine 289 in conjunction with serine 23 results in bidentate 14-3-3 binding, leading to increased Gem protein half-life. Evidence presented shows that phosphorylation of serine 261 is mediated via a cdc42/protein kinase Cζ-dependent pathway. These data demonstrate that phosphorylation of serines 261 and 289, outside the GTP-binding region of Gem, controls its inhibition of Rho kinase β and associated changes in the cytoskeleton.

Sodium and calcium currents of acutely isolated adult rat superior cervical ganglion neurons

Neurons enzymatically isolated from the adult rat superior cervical ganglion (SCG) were investigated using the whole-cell variant of the patch-clamp technique. Currentclamp studies revealed the following mean passive and active membrane properties: resting membrane potential, −54.9 mV; input resistance, 349 MΩ; action potential (AP) threshold, −29.8 mV; AP overshoot, 53.3 mV; AP maximum rate of rise, 166.4 V/s; and AP duration, 3.2 ms. Chemosensitivity to acetylcholine remained intact following enzymatic dispersion. Voltage-clamp studies of a transient tetrodotoxin-sensitive Na+ current revealed activation and inactivation processes which could be fit to modified Boltzmann equations. Na+ current activation parameters for the half activation potential (Vh) and slope factor (K) were −23.3 mV and 5.3 mV, respectively. Inactivation parameters forVh andK were −59.3 mV and 7.6 mV, respectively. Voltage-clamp studies also revealed a high voltageactivated sustained inward current which was eliminated upon removal of external Ca2+, greatly reduced by 500 μM Cd2+, and supported by Ba2+ or Sr2+. Tail current analysis of this Ca2+ current revealed a sigmoidal activation. A low voltage-activated transient Ca2+ current was not observed. We conclude that isolated SCG neurons retain the properties of neurons in intact ganglia and provide several advantages over conventional preparations for the study of voltagegated membrane currents.