Acidosis-stimulated neurons of the medullary raphe are serotonergic.

Neurons of the medullary raphe project widely to respiratory and autonomic nuclei and contain co-localized serotonin, thyrotropin-releasing hormone (TRH), and substance P, three neurotransmitters known to stimulate ventilation. Some medullary raphe neurons are highly sensitive to pH and CO(2) and have been proposed to be central chemoreceptors. Here it was determined whether these chemosensitive neurons are serotonergic. Cells were microdissected from the rat medullary raphe and maintained in primary cell culture for 13-70 days. Immunoreactivity for serotonin, substance P, and TRH was present in these cultures. All acidosis-stimulated neurons (n = 22) were immunoreactive for tryptophan hydroxylase (TpOH-IR), the rate-limiting enzyme for serotonin biosynthesis, whereas all acidosis-inhibited neurons (n = 16) were TpOH-immunonegative. The majority of TpOH-IR medullary raphe neurons (73%) were stimulated by acidosis. The electrophysiological properties of TpOH-IR neurons in culture were similar to those previously reported for serotonergic neurons in vivo and in brain slices. These properties included wide action potentials (4.55 +/- 0.5 ms) with a low variability of the interspike interval, a postspike afterhyperpolarization (AHP) that reversed 25 mV more positive than the Nernst potential for K(+), prominent A current, spike frequency adaptation and a prolonged AHP after a depolarizing pulse. Thus the intrinsic cellular properties of serotonergic neurons were preserved in cell culture, indicating that the results obtained using this in vitro approach are relevant to serotonergic neurons in vivo. These results demonstrate that acidosis-stimulated neurons of the medullary raphe contain serotonin. We propose that serotonergic neurons initiate a homeostatic response to changes in blood CO(2) that includes increased ventilation and modulation of autonomic function.

The kinetics of a non-inactivating K(+) current in alphaT3-1 pituitary gonadotropes is not affected by holding potential.

The non-inactivating K(+) currents in alphaT3-1, a gonadotroph cell line, were recorded in the presence of low intracellular free calcium concentration. The activation kinetics of the whole-cell currents and the gating charge measured from holding potential (V(HOLD)) of -10 mV, V(HOLD)=-80 mV in presence of 4-AP (4-aminopyridine), and V(HOLD)=-10 mV with a hyperpolarizing prepulse to -80 mV were similar. No difference was observed in the onset of currents elicited from the hyperpolarizing potentials, suggesting deviation from the Cole-Moore prediction of increase in the delay of current onset with increasing hyperpolarization. The data suggests that the channel opens with at least one rate-limiting voltage-dependent step, which may imply that the position of the voltage sensor is unaffected by hyperpolarization.

Exploring the architecture of potassium channels using chimaeras to reveal signal transduction.

The chimeric channel, 4N/1, generated from two outwardly rectifying K+ channels by linking the N-terminal cytoplasmic domain of hKv1.4 with the transmembrane body of hKv1.1, functions as an inward rectifier. The operating range of the channel is shifted to hyperpolarizing potentials and it is inactivated at resting membrane potentials. Co-expression of a truncated form of hKv1.1 with the N-terminal domain of hKv1.4 results in the same physiology as the chimaera implying specific interactions between the two segments.

Temperature-dependent conformational changes in a voltage-gated potassium channel.

Temperature was used as a biophysical tool to investigate the energy changes associated with conformational change during the gating of a non-inactivating voltage-gated K+ channel present in the membrane of alpha T3-1 cells, a gonadotroph cell line. The time course of the current activation was described by a single exponential function at three temperatures: 15, 25 and 35 degrees C. The Q10 values were between 1.5 to 1.9 and in agreement with the activation energy determined from Arrhenius plots of the forward and backward rate constants associated with channel opening. The Gibb's free energy change associated with channel opening and closing at various membrane potentials estimated by two approaches yield similar values. The changes in Gibb's free energy (delta G degree) with depolarization potential is a quadratic and more prominent at 15 than at 25 or 35 degrees C. The results suggest that increase in temperature favours movement of voltage sensing segments, and reduces the restraint on them brought about by other parts of the channel molecule.

Transplanting the N-terminus from Kv1.4 to Kv1.1 generates an inwardly rectifying K+ channel.

A chimeric channel, 4N/1, was generated from two outwardly rectifying K+ channels by linking the N-terminal cytoplasmic domain of hKv1.4 (N terminus ball and chain of hKv1.4) with the transmembrane body of hKv1.1 (delta78N1 construct of hKv1.1). The recombinant channel has properties similar to the six transmembrane inward rectifiers and opens on hyperpolarization with a threshold of activation at -90 mV. Outward currents are seen on depolarization provided the channel is first exposed to a hyperpolarizing pulse of -100 mV or more. Hyperpolarization at and beyond -130 mV provides evidence of channel deactivation. Delta78N1 does not show inward currents on hyperpolarization but does open on depolarizing from -80 mV with characteristics similar to native hKv1.1. The outward currents seen in both delta78N1 and 4N/1 inactivate slowly at rates consistent with C-type inactivation. The inward rectification of the 4N/1 chimera is consistent with the inactivation gating mechanism. This implies that the addition of the N-terminus from hKv1.4 to hKv1.1 shifts channel activation to hyperpolarizing potentials. These results suggest a mechanism involving the N-terminal cytoplasmic domain for conversion of outward rectifiers to inward rectifiers.

Voltage gated Na+ channels contribute to membrane voltage fluctuation in alphaT3-1 pituitary gonadotroph cells.

AlphaT3-1 cells showed a slope resistance of 1.8 Gomega. The cell membrane surface was not smooth and a scanning electron micrograph showed a complex structure with blebs and microvilli like projections. The cells showed spontaneous fluctuations at zero current resting membrane potential and hyperpolarization increased the amplitude of membrane potential fluctuations. The amplitude of membrane potential fluctuations at hyperpolarized membrane potential was attenuated on application of TTX to the bath solution. The potential at which half steady state inactivation of isolated sodium current occurred, was at a very hyperpolarized potential (-95.4 mV). The study presented in this paper shows that the voltage gated sodium channels contribute to the increase in the amplitude of electrical noise with hyperpolarization in alphaT3-1 cells.