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These contractions are driven by an intrinsic electrical pacemaker, working through an unknown underlying ionic mechanism that becomes compromised in some forms of lymphedema. In previous studies, T-type voltage-gated Ca<jats:sup>2+<\/jats:sup> channels (VGCCs) were implicated in this pacemaking mechanism, based on the effects of the reputedly selective T-type VGCC inhibitors mibefradil and Ni<jats:sup>2+<\/jats:sup>. Our goal was to test this idea in a more definitive way using genetic knock out mice. First, we demonstrated through both PCR and immunostaining that mouse lymphatic muscle cells expressed Ca<jats:sub>v<\/jats:sub>3.1 and Ca<jats:sub>v<\/jats:sub>3.2 and produced functional T-type VGCC currents when patch clamped. We then employed genetic deletion strategies to selectively test the roles of each T-type VGCC isoform in the regulation of lymphatic pacemaking. Surprisingly, global deletion of either, or both, isoform(s) was without significant effect on either the frequency, amplitude, or fractional pump flow of lymphatic collectors from two different regions of the mouse, studied <jats:italic>ex vivo<\/jats:italic>. Further, both WT and Ca<jats:sub>v<\/jats:sub>3.1<jats:sup>\u2212\/\u2212<\/jats:sup>; 3.2<jats:sup>\u2212\/\u2212<\/jats:sup> double knock-out lymphatic vessels responded similarly to mibefradil and Ni<jats:sup>2+<\/jats:sup>, which substantially reduced contraction amplitudes and slightly increased frequencies at almost all pressures in both strains: a pattern consistent with inhibition of L-type rather than T-type VGCCs. Neither T-type VGCC isoform was required for ACh-induced inhibition of contraction, a mechanism by which those channels in smooth muscle are thought to be targets of endothelium-derived nitric oxide. Sharp intracellular electrode measurements in lymphatic smooth muscle revealed only subtle, but not significant, differences in the resting membrane potential and action potential characteristics between vessels from wild-type and Ca<jats:sub>v<\/jats:sub>3.1<jats:sup>\u2212\/\u2212<\/jats:sup>; 3.2<jats:sup>\u2212\/\u2212<\/jats:sup> double knock-out mice. In contrast, smooth-muscle specific deletion of the L-type VGCC, Ca<jats:sub>v<\/jats:sub>1.2, completely abolished all lymphatic spontaneous contractions. Collectively our results suggest that, although T-type VGCCs are expressed in mouse lymphatic smooth muscle, they do not play a significant role in modulating the frequency of the ionic pacemaker or the amplitude of spontaneous contractions. We conclude that the effects of mibefradil and Ni<jats:sup>2+<\/jats:sup> in other lymphatic preparations are largely or completely explained by off-target effects on L-type VGCCs, which are essential for controlling both the frequency and strength of spontaneous contractions.<\/jats:p>","DOI":"10.1038\/s41598-019-56953-3","type":"journal-article","created":{"date-parts":[[2020,1,9]],"date-time":"2020-01-09T11:02:26Z","timestamp":1578567746000},"update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":60,"title":["T-type, but not L-type, voltage-gated calcium channels are dispensable for lymphatic pacemaking and spontaneous contractions"],"prefix":"10.1038","volume":"10","author":[{"given":"Kim H. 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