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1. HeLa cells were infected with recombinant vaccinia virus containing the T7 RNA polymerase gene and transfected with the cDNA for a rat GABA transporter, GAT1, cloned downstream of a T7 RNA polymerase promoter. Six to sixteen hours after transfection, whole-cell recording with a voltage ramp in the range -90 to 50 mV revealed GABA-induced currents (approximately -100 pA at -60 mV in 100 microM GABA, 16 h after transfection at room temperature). No GABA-induced currents were observed in parental HeLa cells or in mock-transfected cells. 2. GABA-induced currents were suppressed by extracellular perfusion with GABA-free solutions or addition of GAT1 inhibitors SKF89976-A or SKF100330-A. At fixed voltage the GABA dependence of the inward current fitted the Michaelis-Menten equation with a Hill coefficient, n, near unity and an equilibrium constant, K(m), near 3 microM. The Na+ dependence of the inward currents fitted the Michaelis-Menten equation with n approximately equal to 2 and K(m) approximately equal to 10 mM. The constants n and K(m) for GABA and Na+ were independent of voltage in the range -90 to -30 mV. 3. GABA-induced currents reverse direction in the range 5-10 mV. The implication of this result is that GAT1 can mediate electrogenic (electrophoretic) influx or efflux of GABA depending on the membrane voltage. The presence of an outward current in our experiments is consistent with radioactive-labelled flux data from resealed vesicle studies. However, it is inconsistent with frog oocyte expression experiments using the sample clone. In oocytes, GAT1 generates no outward current in a similar voltage range. Smaller intracellular volume or higher turnover rates in the mammalian expression system may explain the outward currents. 4. External GABA induces inward current, and internal GABA induces outward current. However, in cells initially devoid of internal GABA, external GABA can also facilitate an outward current. This GAT1-mediated outward current occurs only after applying negative potentials to the cell. These data are consistent with the concept that negative potentials drive GABA and Na+ into the cell, which then leads to electrogenic efflux through GAT1 at positive voltages. 5. Assuming coupled transport, we estimate the number of transporters, N, times the turnover rate, r, to be Nr approximately 10(9) s-1 under nominal conditions (V = -60 mV, 30 microM GABA, 130 mM Na+ and room temperature). This indicates either very high levels of expression (approximately 10(4) microns-2), assuming published turnover rates (approximately 10 s-1), or turnover rates that are significantly greater than previously reported. As an alternative, a channel may exist in the GAT1 protein that is gated by GABA and Na+ and blocked by GAT1 antagonists. The channel mode of conduction would exist in addition to the coupled, fixed-stoichiometry transporter mode of conduction.

Serotonergic neurons of the medullary raphe nuclei are strongly stimulated by acidosis [1,2], and are putative central respiratory chemoreceptors. We have examined the relationship between serotonergic neurons and blood vessels in the adult rat brain by using immunohistochemistry for tryptophan hydroxylase (TpOH) and injections of arteries with fluorescent albumin in gelatin. Serotonergic neurons within the raphe were closely associated with the basilar artery and its large penetrating branches. There were also homologous serotonergic neurons near large arteries of the rostral and caudal ventrolateral medullary surface (VLMS). We propose that serotonergic neurons on the VLMS contributed to the ventila-tory response to application of acidic solution in classical in vivo experiments that localized the chemoreceptors to that region. However, these neurons may not be specifically sensing CSF pH, but rather the PCO2 of blood in proximal arteries that happen to be located adjacent to the CSF space. In addition, a large number of homologous chemosensitive neurons are also located within the midline raphe nuclei. Perforated patch clamp recordings were made from raphe neurons in tissue culture, and the responses to respiratory acidosis, metabolic acidosis and isohydric hypercapnia were quantified. From a baselin firing rate of 0.2 Hz to 1.5 Hz, an increase in PCO2 from 5% to 9% and a decrease in pHo from 7.4 to 7.17 (at a constant [NaHCO3] of 26 mM) induced a mean increase in firing rate of 1.38 Hz (to 285% of control; n = 11). Metabolic acidosis (5% PCO2, 15 mM [NaHCO3], pHo 7.16) induced a mean increase in firing rate of 1.15 Hz (to 309% of control). Isohydric hypercapnia (9% PCO2, 40 mM [NaHCO3], at constant pHo) induced a mean increase in firing rate of 1.01 Hz (to 384% of control). These neurons also increased their firing rate in response to a decrease in pHo in the absence of CO2 and bicarbonate (in HEPES buffer).The only acid/base change that is shared by these four manipulations is a decrease in pHi, suggesting that this is the primary stimulus for these neurons – as is true for other chemosensitive neurons, including carotid body glomus cells. Using voltage clamp recordings, we have identified a novel ionic current in serotonergic neurons that is highly pH sensitive. This current was activated by calcium influx during depolarization. The major charge carrier was K+ under physiological conditions, but it also had a high permeability to Na+, with a permeability ratio for K+:Na+ of 4:1. It was not blocked by apamin (400 nM), charybdotoxin (240 nM), or TEA (15 mM). The current was extremely sensitive to changes in pH centered near 7.3. The current at pH7.61 was 95% of the maximum current, while the current at pH 6.99 was 2% of the maximum. To our knowledge, a current with these properties has not been reported previously. This current would be predicted to induce changes in membrane potential that have been observed during whole-cell current clamp recordings. Serotonergic medullary raphe neurons are strongly stimulated by a decrease in pHi due to the presence of a novel highly pH-sensitive calcium-activated cation channel. The degree of chemosensitivity of these neurons is large enough to make a major contribution to the changes in ventilation observed in whole animals in response to respiratory acidosis. The location of serotonergic neurons next to blood vessels and their widespread projections suggest that these neurons are chemoreceptors that modulate a variety of brain functions, including but not limited to respiratory control, and thus contribute to pH homeostasis.