(A) Muscle length changes from P7 diaphragm of P2ry1 WT (left) and mutant (right) mice were first recorded in response to HFS in normal or 5 mM [K+]o (black traces), and then recorded in response to 2 bouts of HFS (60 Hz, 45 s) in 10 mM [K+]o (dark and light grey traces = 1st and 2nd HFS, respectively). Peak length changes and fatigue are dramatically affected in P2ry1 mutants by high [K+o]: peak length in response to first HFS in 10 mM [K+]o as a percentage of peak length in response to control HFS in 5 mM [K+]o = 93 ± 20.7 vs. 53.8 ± 24%; n = 3; P2ry1 WT vs. mutant mice, p<0.05; peak length change in response to second HFS in 10 mM [K+]o = 74.2 ± 15.5 vs. 20.9 ± 11% control HFS; n = 3; P2ry1 WT vs. mutant mice; p<0.005; asterisk indicates this almost complete failure to contract; fatigue, or ending length change of second HFS in high 10 mM [K+]o = 41.3 ± 17.8 vs. 7.4 ± 6.8 peak length change of first HFS in 5 mM [K+]o; n = 3; P2ry1 WT vs. mutant mice, p<0.05). (B) Muscle length changes in P2ry1 mutants in response to HFS in lowered [K+]o show a statistically non-significant trend toward less fatigue (ending length change, 2.5 mM [K+]o (grey trace) vs. 5 mM [K+]o (black trace)=75.1 ± 6.2 vs. 83.9 ± 12.9% peak length change; n = 3, p=0.17). (C) Effect of high [K+]o on resting membrane potential (RMP). Representative muscle cell recording before and after (arrowhead) [K+]o was changed from 5 to 10 mM in P7 diaphragm from P2ry1 WT (left) and mutant (right). (D) SD intensity maps of TPSCs in response to HFS (left panels) in P2ry1 WT (upper) and mutant (lower) mice and in response to subsequent treatment with 10 mM [K+]o (right panels). Markedly fewer TPSCs responded to elevated [K+]o in P2ry1 mutants. (E) Peak Ca2+ transient intensities of TPSCs responding to 10 mM [K+]o after 3 bouts of HFS were significantly reduced in P2ry1 mutants or in WT mice treated with the P2Y1R antagonist MRS2500 (18.6 ± 2.5 vs. 14.5 ± 1.5 dB, WT vs. mutant, p<0.05, c > 10 per n; n = 4, Student’s t with Bonferonni correction; 18.6 ± 2.5 vs. 13.8 ± 0.8 dB, WT vs. WT + MRS2500, p<0.01, c > 10 per n; n = 4, Student’s t with Bonferonni correction; fluorescence units from fire CLUT heatmap in SD iu16).