Emerging Concepts in Ion Channel Biophysics

Emerging Concepts in Ion Channel Biophysics

Wednesday Speaker Abstracts

Using Leeches to Discover Novel Ion Channels Involved in Mechanotransduction Elizabeth Heath-Heckman 1 , Maurizio Pellegrino 1 , Diana Bautista 1 , Francisco F. De-Miguel 2 , David Weisblat 1 . 1 University of California - Berkeley, Berkeley, CA, USA, 2 Universidad Nacional Autónoma de México, Mexico City, Mexico, Mexico. Mechanotransduction, mechanisms by which cells convert mechanical stimuli into electrical activity, is a process conserved across all domains of life. Despite its importance, mechanotransduction is not well understood at a molecular level. To better characterize the genes involved, we used leeches in the genus Hirudo whose ventral nerve cord ganglia contain three classes of mechanosensory neurons distinguished by their responses to light touch (T cells), pressure (P), and potentially damaging stimuli (N). To determine which genes confer these behaviors, we performed RNASeq of the above cell types and two non-mechanosensory portions of the ganglion. Some of the most highly regulated transcripts correspond to ion channels already implicated in mechanosensation, such as ASIC, Trp, and CNG. However, two hyperpolarization- activated cyclic nucleotide-gated channels (HCNs) were also upregulated in P and N cells. HCNs, while important in the etiology of chronic pain, have not yet been shown to be involved in mechanosensation. The genome of Helobdella robusta encodes 7 HCNs in what appears to be a lineage-specific gene amplification, suggesting that the roles of HCNs in leeches may differ from, or be more specialized than, those in other animals. In situ hybridization showed at least five of these HCNs are expressed in the nerve cord ganglia in juvenile animals, one of them identical to the enrichment found in Hirudo . Preliminary experiments suggest that RNAi of the upregulated HCN in Hirudo P cells abrogates the normal hyperpolarization-induced “sag” current, suggesting it is the primary HCN in these cells. Future experiments will include using CRISPR-Cas9 to “knock-out” HCN genes in Helobdella and determine the effect on mechanosensation, as well as using Hirudo ex vivo ganglion preparations and primary cell culture to determine how the loss of HCN activity changes their response to mechanical stimuli.

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