Molecular and functional characterization of the electroneutral Na/HCO3 cotransporter NBCn1 in rat hippocampal neurons.

We examined molecular and electrophysiological properties of the electroneutral sodium/bicarbonate cotransporter (NBCn1) that is present in rat hippocampal neurons. By PCR, a deletion variant (NBCn1-E) that lacks 123 amino acids in the cytoplasmic N-terminal domain was found in adult neurons. The previously characterized NBCn1-B, which does not have the deletion, was detected in embryonic neurons. In Xenopus oocytes, NBCn1-E raised the intracellular pH in the presence of HCO(3) without significantly affecting the membrane potential. Despite this electroneutral cotransport activity, the transporter mediated a steady-state current that positively shifted the resting potential by almost 30 mV. The mean reversal potential of the steady-state current was -21.2 mV, close to the resting potential of -21.4 mV. The reversal potential shifted 26 mV in response to a 10-fold increase of external Na(+) for concentrations above 10 mm. The current activity mediated by the transporter was unaffected by K(+), Mg(2+), Ca(2+), or Cl(-). Stable expression of NBCn1-E in human embryonic kidney cells also evoked an inward current that shifted the resting potentials more positive compared with the sham-transfected controls. In primary cultures of embryonic hippocampal neurons, the NBCn1 protein was localized in somatodendrites and synapses. NBCn1 protein was partially colocalized with the postsynaptic density protein PSD-95. Single-cell PCR showed that NBCn1 mRNA expression was present in both gamma-aminobutyric acid (GABA)ergic and non-GABAergic neurons. We propose that NBCn1 in hippocampal neurons may affect neuronal activity by regulating local pH as well as steady-state inward currents at synapses.

Statistical mechanics model for the interaction between the neurotransmitter gamma-aminobutyric acid and GABAA receptors.

Interactions between the neurotransmitter gamma-aminobutyric acid (GABA) and GABAA receptor ion channels play an important role in the central nervous system. A statistical mechanics model is proposed for the interaction between GABA and GABAA receptors. The model provides good fits to the electrophysiology data as well as an estimation of receptor activation energies, and predicts the temperature dependence consistent with measurements. In addition, the model provides insights into single channel conductance measurements. This model is also applicable to other ligand-gated ion channels with similar pentameric structures.

Characterization of an amiloride binding region in the alpha-subunit of ENaC.

One of the defining characteristics of the epithelial sodium channel (ENaC) is its block by the diuretic amiloride. This study investigates the role of the extracellular loop of the alpha-subunit of ENaC in amiloride binding and stabilization. Mutations were generated in a region of the extracellular loop, residues 278-283. Deletion of this region, WYRFHY, resulted in a loss of amiloride binding to the channel. Channels formed from wild-type alpha-subunits or alpha-subunits containing point mutations in this region were examined and compared at the single-channel level. The open probabilities (Po) of wild-type channels were distributed into two populations: one with a high Po and one with a low Po. The mean open times of all the mutant channels were shorter than the mean open time of the wild-type (high-Po) channel. Besides mutations Y279A and H282D, which had amiloride binding affinities similar to that of wild-type alpha-ENaC, all other mutations in this region caused changes in the amiloride binding affinity of the channels compared with the wild-type channel. These data provide new insight into the relative position of the extracellular loop with respect to the pore of ENaC and its role in amiloride binding and channel gating.