Geoffrey G. Schofield

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.

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.

Neuropeptide Y blocks a calcium current in C cells of bullfrog sympathetic ganglia.

The effects of neuropeptide Y (NPY) on the Ca2+ current of enzymatically dispersed neuronal somata from bullfrog paravertebral sympathetic ganglia were investigated using the patch-clamp technique. In C neurons, NPY induced a concentration-dependent decrease in the amplitude of the Ca2+ current and induced a slow biphasic current rising phase. Upon removal of NPY these parameters returned to near control values. NPY had no discernable effect on the Ca2+ current recorded in B neurons.

Potassium currents of acutely isolated adult rat superior cervical ganglion neurons

K+ currents of adult rat superior cervical ganglion neurons were studied using the voltage-clamp technique. Neuronal somata were dissociated from the ganglion using an enzymatic dispersion technique and voltage-clamped using the whole-cell patch-clamp technique. In solutions designed to isolate K+ currents, depolarization from a prepulse potential of -100 mV induced both transient and sustained outward current components. The transient current was completely eliminated by depolarization to -50 mV. The remaining sustained current component could be separated further into Ca2+-sensitive and Ca2+-insensitive components by superfusion with a Ca2+-free external solution. The transient current, which could be isolated by digital subtraction, rose rapidly and decayed over the subsequent 80 ms. Reversal potential determinations in different K+-containing solutions demonstrated that the current was carried primarily by K+. The transient current showed voltage-dependent inactivation, showing 50% inactivation near -87 mV and was completely inactivated at potentials more positive than -60 mV. The transient current recovered from inactivation with a voltage-dependent time course, the time course of inactivation decreasing with hyperpolarization. This transient outward current had characteristics of IA. The sustained Ca2+-insensitive outward current showed little decay over 800 ms and was also carried primarily by K+. This current component had characteristics similar to the delayed rectifier. A third sustained outward current eliminated by superfusion with Ca2+-free external solution had characteristics similar to the Ca2+-dependent K+ current.

Somatostatin blocks a calcium current in acutely isolated adult rat superior cervical ganglion neurons.

Somatostatin-like immunoreactivity has been reported to occur in the postganglionic neurons of sympathetic ganglia. We therefore investigated the effect of somatostatin (SOM) on the Ca2+ current in sympathetic neurons. Voltage-clamp recordings, using the whole-cell patch-clamp technique, were made from acutely isolated adult rat superior cervical ganglion (SCG) neurons in solutions (external and internal) designed to isolate Ca2+ currents. Application of 0.001-1.0 microM [D-Trp8]SOM resulted in a rapid, reversible and concentration-dependent decrease in the amplitude of the Ca2+ current evoked from a holding potential of -80 mV. The concentration-response relationship for SOM could be fitted to a single-site binding model with an apparent dissociation constant of 11 nM; the maximal attainable block of Ca2+ current by SOM was 50%. SOM also produced a pronounced slowing of the Ca2+ current rising phase, especially at more depolarized potentials. At higher concentrations (0.03-1.0 microM), prolonged application of SOM resulted in a progressive decrease in blocking ability. The results are consistent with a neurotransmitter and/or neuromodulator role for SOM in the sympathetic nervous system.

Somatostatin cyclic octapeptide analogs which preferentially bind to SOMa receptors block a calcium current in rat superior cervical ganglion neurons

To characterize further the somastatin (SOM) receptor mediating Ca2+ current reduction in rat superior cervical ganglion (SCG) neurons, the effects of three synthetic SOM octapeptide analogs, D-Phe-Cys-Tyr-D-Trp-Lys-Val-Cys-Thr-NH2 (IM-4-82), D-Nal-Cys-Tyr-D-Trp-Lys-Val-Cys-Thr-NH2 (DC 13-116), and D-Phe-Cys-Phe-D-Trp-Lys-Thr-Cys-Thr-OL (SMS 201-995), which bind preferentially to pituitary SOM receptors (SOMa) were investigated. Ca2+ currents were recorded using the whole-cell variant of the patch-clamp technique from neurons isolated enzymatically from adult rat SCG. Application of the SOM analogs (0.003-3 microM) produced a rapid, reversible, and concentration-dependent decrease in Ca2+ current amplitude in addition to slowing the rising phase of the Ca2+ current. Estimates of the concentration producing half-maximal block (EC50) and maximum attainable block (Bmax) for DC 13-116, IM 4-28, and SMS 201-995 were 196, 67, and 9.5 nM, respectively, and 52, 57, and 48%, respectively. The results suggest that the SOM receptor on SCG neurons more closely resembles the SOMa receptor of the anterior pituitary than the SOMb receptor of cerebral cortical membranes.

The effect of pumiliotoxin-B on the excitability of bullfrog sympathetic neurons

The effects of the alkaloid pumiliotoxin-B were investigated on neurons from bullfrog paravertebral ganglia using current-clamp techniques. Pumiliotoxin-B (2 microM) induced repetitive action potential discharge or bursting pacemaker activity in response to a single stimulus. The toxin had no significant effect on the mean resting potential or action potential characteristics of single action potentials evoked prior to the action potential discharge onset. During the action potential discharge, action potential threshold and afterhyperpolarization amplitude were decreased. In the presence of pumiliotoxin-B, single action potentials were followed by a depolarizing afterpotential. Pumiliotoxin-B still induced action potential discharge in Ca2+-free or Cd2+-containing solutions. Brief superfusion with a Na+-free or tetrodotoxin-containing solution abolished the pumiliotoxin-B-induced action potential discharge prior to the blockade of directly elicited single action potentials. These solutions decreased or abolished the depolarizing afterpotential. Pumiliotoxin-B increases membrane excitability and can induce a stimulation-dependent action potential discharge which appears to result from a tetrodotoxin-sensitive Na+-sensitive potential.

Single acetylcholine channel currents in sympathetic neurons

Single acetylcholine (ACh) channel currents were studied by the gigaohm patch-clamp technique in cultured sympathetic neurons of the bullfrog, Rana catesbeiana. Recordings were made at 22 degrees C on cell-attached and excised membrane patches. When ACh (0.5-1 microM) was present in the pipette, a single class of inward currents was observed with a chord conductance of 30 pS and a reversal potential of -2 mV. The mean channel open time was 11.6 ms at -65 mV and showed little or no voltage-dependence over the range -85 to -45 mV. These channels appear to mediate the fast nicotinic excitatory postsynaptic current.

Properties of Wild-Type and Fluorescent Protein-Tagged Mouse Tetrodotoxin-Resistant Sodium Channel (Nav1.8) Heterologously Expressed in Rat Sympathetic Neurons

The tetrodotoxin (TTX)-resistant Na(+) current arising from Na(V)1.8-containing channels participates in nociceptive pathways but is difficult to functionally express in traditional heterologous systems. Here, we show that injection of cDNA encoding mouse Na(V)1.8 into the nuclei of rat superior cervical ganglion (SCG) neurons results in TTX-resistant Na(+) currents with amplitudes equal to or exceeding the currents arising from natively expressing channels of mouse dorsal root ganglion (DRG) neurons. The activation and inactivation properties of the heterologously expressed Na(V)1.8 Na(+) channels were similar but not identical to native TTX-resistant channels. Most notably, the half-activation potential of the heterologously expressed Na(V)1.8 channels was shifted about 10 mV toward more depolarized potentials. Fusion of fluorescent proteins to the N- or C-termini of Na(V)1.8 did not substantially affect functional expression in SCG neurons. Unexpectedly, fluorescence was not concentrated at the plasma membrane but found throughout the interior of the neuron in a granular pattern. A similar expression pattern was observed in nodose ganglion neurons expressing the tagged channels. In contrast, expression of tagged Na(V)1.8 in HeLa cells revealed a fluorescence pattern consistent with sequestration in the endoplasmic reticulum, thus providing a basis for poor functional expression in clonal cell lines. Our results establish SCG neurons as a favorable surrogate for the expression and study of molecularly defined Na(V)1.8-containing channels. The data also indicate that unidentified factors may be required for the efficient functional expression of Na(V)1.8 with a biophysical phenotype identical to that found in sensory neurons.