Differential distribution and function of hyperpolarization-activated channels in sensory neurons and mechanosensitive fibers.

Sensory neurons express hyperpolarization-activated currents (I(H)) that differ in magnitude and kinetics within the populations. We investigated the structural basis for these differences and explored the functional role of the I(H) channels in sensory neurons isolated from rat nodose ganglia. Immunohistochemical studies demonstrated a differential distribution of hyperpolarization-activated cyclic nucleotide-gated (HCN) protein (HCN1, HCN2, HCN4) in sensory neurons and peripheral terminals. HCN2 and HCN4 immunoreactivity was present in all nodose neurons. In contrast, only 20% of the total population expressed HCN1 immunoreactivity. HCN1 did not colocalize with IB4 (a marker for C-type neurons), and only 15% of HCN1-positive neurons colocalized with immunoreactivity for the vanilloid receptor VR1, another protein associated primarily with C-type neurons. Therefore, most HCN1-containing neurons were A-type neurons. In further support, HCN1 was present in the mechanosensitive terminals of myelinated but not unmyelinated sensory fibers, whereas HCN2 and HCN4 were present in receptor terminals of both myelinated and unmyelinated fibers. In voltage-clamp studies, cell permeant cAMP analogs shifted the activation curve for I(H) to depolarized potentials in C-type neurons but not A-type neurons. In current-clamp recording, CsCl, which inhibits only I(H) in nodose neurons, hyperpolarized the resting membrane potential from -63 +/- 1 to -73 +/- 2 mV and nearly doubled the input resistance from 1.3 to 2.2 GOmega. In addition, action potentials were initiated at lower depolarizing current injections in the presence of CsCl. At the sensory receptor terminal, CsCl decreased the threshold pressure for initiation of mechanoreceptor discharge. Therefore, elimination of the I(H) increases excitability of both the soma and the peripheral sensory terminals.

Potassium channels Kv1.1, Kv1.2 and Kv1.6 influence excitability of rat visceral sensory neurons.

Voltage-gated potassium channels, Kv1.1, Kv1.2 and Kv1.6, were identified as PCR products from mRNA prepared from nodose ganglia. Immunocytochemical studies demonstrated expression of the proteins in all neurons from ganglia of neonatal animals (postnatal days 0-3) and in 85-90 % of the neurons from older animals (postnatal days 21-60). In voltage clamp studies, alpha-dendrotoxin (alpha-DTX), a toxin with high specificity for these members of the Kv1 family, was used to examine their contribution to K(+) currents of the sensory neurons. alpha-DTX blocked current in both A- and C-type neurons. The current had characteristics of a delayed rectifier with activation positive to -50 mV and little inactivation during 250 ms pulses. In current-clamp experiments alpha-DTX, used to eliminate the current, had no effect on resting membrane potential and only small effects on the amplitude and duration of the action potential of A- and C-type neurons. However, there were prominent effects on excitability. alpha-DTX lowered the threshold for initiation of discharge in response to depolarizing current steps, reduced spike after-hyperpolarization and increased the frequency/pattern of discharge of A- and C-type neurons at membrane potentials above threshold. Model simulations were consistent with these experimental results and demonstrated how the other major K(+) currents function in response to the loss of the alpha-DTX-sensitive current to effect these changes in action potential wave shape and discharge.

P2X purinergic receptor channel expression and function in bovine aortic endothelium.

We examined bovine aortic endothelial cells (BAECs) for the functional expression of P2X receptors, the ATP-gated cation channels. We identified the P2X subtypes present in BAECs using RT-PCR. mRNA was present for only three of seven family members: P2X4, P2X5, and P2X7. We then characterized agonist-activated currents in whole cell and outside-out patch recordings using 2-methyl-thio-ATP (MeSATP) as a P2X4 and P2X5 receptor agonist and 2',3'-O-(4-benzoylbenzoyl)ATP (BzATP) as a P2X7 receptor agonist. MeSATP (10-20 microM) produced current with characteristics of P2X4 receptors. The current was an inwardly rectifying current, reversed near 0 mV, slowly desensitized, was not blocked by suramin (300 microM) or reactive blue (60 microM), and had a single channel conductance of 36 pS. BzATP (10-100 microM), on the other hand, activated a 9-pS channel with sustained activity in the continued presence of the agonist. BzATP-activated current was blocked by reactive blue (60 microM) and by suramin (approximately 50% block at 300 microM). We confirmed, by immunocytochemistry, the presence of P2X4 and P2X7 protein. The agonists failed, however, to induce significant uptake of the large molecule YO-PRO, indicating the lack of pore development that has been demonstrated for P2X7 and P2X4 in response to agonist in some cell types.