Volume 127, Issue 6, Pages 1739-1747 (December 2004) The ion channel ASIC1 contributes to visceral but not cutaneous mechanoreceptor function  Amanda J. Page, Stuart M. Brierley, Christopher M. Martin, Carlos Martinez-Salgado, John A. Wemmie, Timothy J. Brennan, Erin Symonds, Taher Omari, Gary R. Lewin, Michael J. Welsh, L. Ashley Blackshaw  Gastroenterology  Volume 127, Issue 6, Pages 1739-1747 (December 2004) DOI: 10.1053/j.gastro.2004.08.061 Copyright © 2004 American Gastroenterological Association Terms and Conditions

Figure 1 Diagram of recording set-up. Features of both colonic and gastroesophageal recording equipment are shown and major differences are highlighted. Gastroenterology 2004 127, 1739-1747DOI: (10.1053/j.gastro.2004.08.061) Copyright © 2004 American Gastroenterological Association Terms and Conditions

Figure 2 Expression of ASIC1 in sensory ganglia. (A) PCR analysis of messenger RNA expression of ASIC1 variants in mouse nodose and DRG, with negative control (no RT). A product of expected size was detected for both variants in ASIC1+/+ mice. ASIC1b messenger RNA still could be detected in ASIC1−/− mice. (B) Immunoprecipitation and Western blot of protein with a pan-ASIC1 antibody either from COS cells transfected with recombinant ASIC1b or 1a, or from DRG taken from ASIC1+/+ and −/− mice. ASIC1 protein was evident in transfected cells and in ASIC1+/+ DRG, but not in −/− DRG. Gastroenterology 2004 127, 1739-1747DOI: (10.1053/j.gastro.2004.08.061) Copyright © 2004 American Gastroenterological Association Terms and Conditions

Figure 3 Disrupting ASIC1 alters the properties of colonic and gastroesophageal mechanoreceptors and slows gastric emptying. (A) (i) Stimulus-response function of colonic serosal/mesenteric mechanoreceptors from ASIC1+/+ (•, n = 26, N = 17) and −/− mice (□, n = 32, N = 25), showing a significant increase in the stimulus response function in ASIC1−/− mice (P < .001; 2-way ANOVA). (ii) Adaptation of colonic serosal/mesenteric mechanoreceptors from ASIC1+/+ and −/− mice. Adaptation of colonic mechanoreceptors in ASIC1−/− mice showed significantly steeper slope than in ASIC1+/+ mice (P < .001, linear regression), indicating more rapid adaptation overall. The adaptation profile also was altered significantly (P < .001; 2-way ANOVA). Wild-type afferents adapted rapidly initially (200–400 ms), and then more slowly over the remainder of the response (*P < .05; Bonferroni post hoc test, data from 1000-mg stimulus). (B) (i) Stimulus-response function of vagal tension receptors from ASIC1+/+ (•, n = 40, N = 23) and −/− mice (□, n = 47, N = 30), showing a significant increase in the stimulus response function in ASIC1−/− mice (P < .01; 2-way ANOVA). (ii) Adaptation of vagal tension receptors to maintain stretch over 1 minute, with spikes grouped into 10-second bins. No significant difference was seen in adaptation between ASIC1+/+ and −/− at any point of the response. (iii) Stimulus-response function of vagal mucosal mechanoreceptors from ASIC1+/+ (•, n = 17, N = 11) and −/− mice (□, n = 28, N = 13), showing a significant increase in the stimulus-response function in ASIC1−/− mice (P < .05; 2-way ANOVA). (C) Gastric half emptying time of a solid meal in ASIC1+/+ (N = 8) and −/− mice (N = 14). ASIC1−/− mice had a significantly slower gastric emptying rate compared with +/+ (*P = .01; unpaired t test). Gastroenterology 2004 127, 1739-1747DOI: (10.1053/j.gastro.2004.08.061) Copyright © 2004 American Gastroenterological Association Terms and Conditions

Figure 4 Disrupting ASIC1 has no effect on the sensitivity of cutaneous mechanoreceptors or on behavioral responses to somatic stimuli. (A) Stimulus-response function of cutaneous primary afferents to applied displacements (i) rapidly adapting mechanoreceptor fibers, ASIC1+/+, n = 27, ASIC1−/−, n = 52; (ii) slowly adapting mechanoreceptor fibers, ASIC1+/+, n = 51, ASIC1−/−, n = 80; (iii) slowly conducting myelinated mechanonociceptors (A-fiber mechanonociceptors), ASIC1+/+, n = 52, ASIC1−/−, n = 54; (iv) down-hair afferents, ASIC1+/+, n = 33, ASIC1−/−, n = 45; and (v) C-fiber nociceptors, ASIC1+/+, n = 18, ASIC1−/−, n = 24. •, ASIC1+/+ fibers; □, −/− fibers. No significant difference in sensitivity was found between the 2 groups as assessed by 2-way ANOVA. ASIC1+/+, N = 27; ASIC1−/−, N = 28. (B) (i) Paw withdrawal latency to thermal stimulation with radiant heat (ASIC1+/+, N = 16; −/−, N = 17). No significant difference in paw withdrawal latency was observed between the 2 groups. (ii) Frequency of paw withdrawal to mechanical stimulation with von Frey monofilaments (ASIC1+/+, N = 5; −/−, N = 9). No significant difference in response frequency was observed between the 2 genotypes. Gastroenterology 2004 127, 1739-1747DOI: (10.1053/j.gastro.2004.08.061) Copyright © 2004 American Gastroenterological Association Terms and Conditions