Molecular identity, ontogeny, and cAMP modulation of the hyperpolarization-activated current in vestibular ganglion neurons

Abstract

Properties, developmental regulation, and cAMP modulation of the hyperpolarization-activated current (I(h)) were investigated by the whole cell patch-clamp technique in vestibular ganglion neurons of the rat at two postnatal stages (P7-10 and P25-28). In addition, by RT-PCR and immunohistochemistry the identity and distribution of hyperpolarization-activated and cyclic nucleotide-gated channel (HCN) isoforms that generate I(h) were investigated. I(h) current density was larger in P25-28 than P7-10 rats, increasing 410% for small cells (<30 pF) and 200% for larger cells (>30 pF). The half-maximum activation voltage (V(1/2)) of I(h) was -102 mV in P7-10 rats and in P25-28 rats shifted 7 mV toward positive voltages. At both ages, intracellular cAMP increased I(h) current density, decreased its activation time constant (τ), and resulted in a rightward shift of V(1/2) by 9 mV. Perfusion of 8-BrcAMP increased I(h) amplitude and speed up its activation kinetics. I(h) was blocked by Cs(+), zatebradine, and ZD7288. As expected, these drugs also reduced the voltage sag caused with hyperpolarizing pulses and prevented the postpulse action potential generation without changes in the resting potential. RT-PCR analysis showed that HCN1 and HCN2 subunits were predominantly amplified in vestibular ganglia and end organs and HCN3 and HCN4 to a lesser extent. Immunohistochemistry showed that the four HCN subunits were differentially expressed (HCN1 > HCN2 > HCN3 ≥ HCN4) in ganglion slices and in cultured neurons at both P7-10 and P25-28 stages. Developmental changes shifted V(1/2) of I(h) closer to the resting membrane potential, increasing its functional role. Modulation of I(h) by cAMP-mediated signaling pathway constitutes a potentially relevant control mechanism for the modulation of afferent neuron discharge.