Comparative Biochemistry and Physiology Part B: Biochemistry and Molecular Biology
A potential role for hyperpolarization-activated cyclic nucleotide-gated sodium/potassium channels (HCNs) in teleost acid-base and ammonia regulation
Graphical abstract
Introduction
Hyperpolarization-activated cyclic nucleotide-gated sodium/potassium channels (HCNs) belong to the voltage-gated cation channel superfamily and are integral membrane proteins that contribute to the generation of membrane potentials in excitable and non-excitable cells (Jackson et al., 2007). In mammals, the four different isoforms / paralogs (HCN1 to HCN4) have been identified to be very similar in their structure: Four subunits are believed to form a tetramer around a core with each subunit containing 6 transmembrane domains hosting a voltage sensor domain in segment 4 and a cation conducting pore between segments 5 and 6 (Jackson et al., 2007). Despite their structural similarities, HCNs can be distinguished by their different quantitative electrochemical properties including voltage-dependence, activation/deactivation kinetics and sensitivity to the nucleotide cyclic AMP (cAMP)(Biel et al., 2009; Santoro and Tibbs, 1990). HCNs contribute to the generation of a current commonly referred to as the “funny” current If or Ih. With a Nernst potential of -20 mV, If/Ih is inwardly directed at rest and depolarizes the cellular membrane potential. In contrast to being activated upon membrane depolarization like the majority of cellular conductances, If / Ih is rather activated by hyperpolarization to membrane potentials more negative than −55 mV (Biel et al., 2009). HCNs in fishes exhibit very similar properties to the mammalian HCNs, however, they are present in multiple isoforms which likely arose from the most ancestral HCN3 during gene duplication events (Jackson et al., 2007).
Predominantly, HCNs are known to contribute to the spontaneous beating in pacemaker cells and hence for their role in cardiac pacemaking, and/or as key participants in nerve signal transduction, in both mammals (Biel et al., 2009) and fishes (Hassinen et al., 2017; Jackson et al., 2007; Sutcliff et al., 2020; Wilson et al., 2013). Carrisoza-Gaytán and colleagues (Carrisoza-Gaytán et al., 2011), however, discovered that HCN2 in the rat was also involved in renal acid-base regulation by promoting NH4+ transport, specifically in acid-secreting intercalated cells in the distal nephron. Furthermore, an HCN channel has been implicated in branchial acid-base regulation in the green crab, Carcinus maenas. HCN in this crab also appears to promote transport of NH4+, probably by substitution of NH4+ for K+ due to its almost identical size and charge (Fehsenfeld and Weihrauch, 2016; Towle and Holleland, 1987). This leaves fish as particularly interesting study animals, especially for HCN2, as they possess both gills and kidneys that are involved in acid-base and ammonia regulation to different extents under different physiological circumstances (Fehsenfeld et al., 2019; Fehsenfeld and Wood, 2018; Perry and Gilmour, 2006; Wright and Wood, 2012).
As multiple isoforms and paralogs of HCNs are present in other teleosts (Jackson et al., 2007), we hypothesized here that (1) different isoforms of HCNs in the goldfish (Carassius auratus) would be differentially expressed in excretory organs and especially in gills and kidney, and (2) acid-base and ammonia disturbances would result in significant changes in mRNA abundance of these isoforms. In order to test our hypotheses, we partially cloned HCN paralogs initially identified in the Cyprinus carpio transcriptome and later verified in the goldfish genome using known mammalian and other teleost sequences. We then used quantitative real-time PCR (qPCR) to assess their mRNA abundance in response to challenges of acid-base/ammonia homeostasis by feeding (which induces a postprandial “acidic tide” as well as an ammonia load in agastric fish such as goldfish; Fehsenfeld and Wood, 2018; Wood, 2019; Wood et al., 2010) and exposure to high environmental ammonia. We also checked for simultaneous changes in the mRNA expression of Rhesus glycoproteins (ammonia channels) which are known to be sensitive to these treatments in gills and kidney (e.g. Fehsenfeld and Wood, 2018; Nawata et al., 2007; Sinha et al., 2013; Zhang et al., 2015; Zimmer et al., 2010).
Section snippets
Animal care
Goldfish C. auratus with an approximate length of ~5 cm (2.3 ± 0.1 g, mean ± SE, N = 18) were obtained from Noah's Pet Ark (Vancouver, BC, Canada) and kept in recirculating and filtered 75-L aquaria in dechlorinated Vancouver tap water [in μmol/L: ~70 Na+, 73 Cl−, 7 Mg2+, 89 Ca2+, pH 6.94 ± 0.03] at 20 °C at a light cycle of 12:12-h light-dark at a maximum density of 1 animal/3 L. Water ammonia and pH levels were closely monitored, and water changes were performed 1–2 times per week as
Identification of CaHCN isoforms
Overall, in the Cyprinus carpio transcriptome, we identified two entries in the NCBI database for potential HCN1-like (GeneBank accession numbers XM019082034.1, XM019116212.1), seven entries annotated for HCN2-like (XM019071468.1, XM019101656.1, XM019105399.1, XM019118763.1, XM019119676.1/XM019119677.1, XM019122180.1), five entries annotated for HCN3-like (XM019068612.1/XM019068613.1, XM019088348.1, XM019099869.1, XM019125329.1) and three entries annotated for HCN4-like (XM019116252.1,
Phylogeny of CaHCN
In the present study, we were able to identify six C. auratus HCN paralogs (CaHCN1, CaHCN2a, CaHCN2b, CaHCN3, CaHCN4a, CaHCN4b), all of which were present as two distinguishable isoforms. The phylogenetic analysis of the identified goldfish paralogs supports HCN3 to be the most ancient as also suggested by Jackson and colleagues (Jackson et al., 2007), and hence it might have served as the “template” for the duplication event. Interestingly, and in contrast to hagfishes (Wilson et al., 2013) or
Conclusion
Despite the known ability of HCN channels to transport NH4+, and their well-documented pH-sensitivity, their potential contributions to more general physiological functions (other than to cardiac pacemaking and nerve conduction) have received relatively little investigation. The present study shows that mRNA abundance of specific HCN paralogs significantly changed in goldfish in response to an either internal (feeding) or external (high environmental ammonia) acid-base and/or ammonia
Author contributions
SF and CMW conceived the study. All experiments and analyses were performed by SF. SF wrote the original draft, while CMW reviewed and edited the manuscript.
Funding
This work was supported by a Discovery Grant from the Natural Sciences and Engineering Research Council of Canada to CMW (NSERC PIN-2017-03843).
Declaration of Competing Interest
The authors declare no competing or financial interests.
Acknowledgements
The authors thank Rachel Sutcliff (UBC) for sharing fish HCN sequences and contigs.
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