The Cloning And Characterization Of Two Urochordate Hyperpolarization-activated Cyclic Nucleotide-modulated (HCN) Channels

Abstract

We have cloned two novel HCN genes from the urochordate species, Ciona Intestinalis, referred to as ciHCNa and ciHCNb, which share ∼50% identity with mammalian HCN isoforms between S1 and the end of the CNBD. Based on our previous phyogenetic analysis of primary sequence, ciHCNb is more closely related to the vertebrate isoforms except that it lacks a putative N-glycosylation site near the pore, which is found in vertebrate sequences and ciHCNa. When expressed in Xenopus oocytes, both clones produce a slowly-activating current (Ih) in response to hyperpolarizing pulses, which is inhibited by Cs+ and ZD7288. For ciHCNb, Ih has a reversal potential of -32.4mV in 5mM K+/91 mM Na+ extracellular solution, which shifts to -1.9mV in 96mM K+ solution. This suggests that Ih is carried by both Na+ and K+, a characteristic of other known HCN channels. Fitting Ih traces, generated from a -70 mV pulse, with a single exponential function yielded values for τ of 1.15 +/- 0.06s and 1.14 +/- 0.17s for ciHCNa and ciHCNb, respectively. Application of 10mM 8-bromo cAMP in the 96mM K+ bath solution produced positive shifts in the Ih activation curve for each clone. Boltzmann fits of normalized tail current amplitudes versus test voltage (Ih activation curves) yielded V1/2 values in the presence of cAMP of -59.86 +/- 1.62mV and -48.5 +/- 3.70mV for ciHCNa and ciHCNb, respectively. ciHCNb also produces a very large instantaneous current, which is blocked by Cs+ and ZD7288 and proportional in size to Ih. This component more similar in size to that of the sea urchin SPIH channel than to those of the mammalian HCNs. Together, the data suggest that the mammalian HCNs are evolutionarily closer to ciHCNa than to ciHCNb.