Asymmetric Divergence in Structure and Function of HCN Channel Duplicates in Ciona intestinalis

Abstract

Hyperpolarization-activated Cyclic Nucleotide (HCN) channels are voltage-gated cation channels and are critical for regulation of membrane potential in electrically active cells. To understand the evolution of these channels at the molecular level, we cloned and examined two of three HCN homologs of the urochordate Ciona intestinalis (ciHCNa and ciHCNb). ciHCNa is like mammalian HCNs in that it possesses similar electrical function and undergoes N-glycosylation of a sequon near the pore. ciHCNb lacks the pore-associated N-glycosylation sequon and is predictably not N-glycosylated, and it also has an unusual gating phenotype in which the channel's voltage-sensitive gate appears to close incompletely. Together with previous findings, the data support an evolutionary trajectory in which an HCN ancestor underwent lineage-specific duplication in Ciona, to yield one HCN with most features that are conserved with the mammalian HCNs and another HCN that has been uniquely altered.

A Mechanism for the Auto-inhibition of Hyperpolarization-activated Cyclic Nucleotide-gated (HCN) Channel Opening and Its Relief by cAMP

“…The protein was expressed in deuterated M9 media, and 1 h prior to induction, 100 mg of methyl-13 C-␣-ketoisovaleric acid and 100 mg of methyl 13 C-␣-ketobutyric acid were added to the media. The protein was concentrated to 250 M in 25 mM phosphate buffer, pH 6.5, 100 mM KCl, 99% 2 H 2 O.…”

“…The mixing times were 200 and 300 ms. The NOESY-15 N HSQC and 15 N-HSQC-NOESY- 15 N HSQC experiments (30) were acquired using uniformly 13 C 15 N-labeled HCN4(579 -707). The mixing time was 130 ms.…”

“…For this purpose, we utilized an HCN4 construct, i.e. human HCN4(563-724), that is stabilized in the monomeric state through the deletion of the AЈ, BЈ-helices required for tetramerization (11,13,21). This HCN4 segment spans the linker helices CЈ-FЈ as well as the CBD and is referred to here as the "extended CBD" or "eCBD" (Fig.…”

Functional Characterization of Cnidarian HCN Channels Points to an Early Evolution of Ih

“…Thus the molecular basis for differential cyclic nucleotide sensitivity in NvHCN1 and NvHCN2 could lie outside the binding region. 8-Br-cAMP has been used extensively to explore the cAMP sensitivity of HCN channels [ 49 , 52 , 82 ] and typically shows close correspondence with results obtained by direct application of cyclic nucleotides to the intracellular face of excised patches [ 75 , 83 , 84 ]. Nevertheless, it remains possible the cAMP modulation we observed for NvHCN2 is indirect because we tested cyclic nucleotide sensitivity by bath application of 8-Br-cAMP.…”

“…Mammals have 4 HCN channel orthologs with diverse gating properties [ 46 – 48 ], and HCN channels encoding hyperpolarization-gated I h currents have been characterized in tunicates [ 49 ], sea urchin [ 50 ], honey bee [ 51 ] and lobster [ 52 ]. Classical HCN channels are thus widespread in the Bilateria, although they appear to have been lost in nematode C .…”