Elementary TRPV4 sparklets are produced by Ca²⁺ influx via single TRPV4 channels. (A) TIRF image of three sparklet sites in a representative tsA-201 cell expressing TRPV4 channels. The traces to the right of the image show time courses of [Ca²⁺]_i in the region of the cell marked by the green circle before and after the application of 10 nM GSK. The broken red line marks the amplitude of quantal TRPV4 sparklets. The solid red line highlights basal [Ca²⁺]_i. (B) All-points histogram of TRPV4 sparklets after GSK application. The red line is the best fit to the data using a multi-Gaussian function with a q value of 48 nM. (C) TIRF image of a representative arterial myocyte and time courses of [Ca²⁺]_i within the area of the cell demarcated by the green circle before and after exposure to 100 nM GSK. (D) Histogram of the amplitudes of TRPV4 sparklets before (white bars) and after (gray bars) GSK application. Data were fit (solid lines) with a multi-Gaussian function (q = 48 nM). (E) Scatter plot of TRPV4 sparklet activity in arterial myocytes. The red lines are the median values. *, P < 0.05; Mann-Whitney test. (F) Plot of the relationship between TRPV4 sparklet signal mass and ΔQ_TRPV4 in arterial myocytes. The solid line is a linear fit to the data (r² = 0.97). The inset shows a simultaneous recording of a TRPV4 current (black) and sparklet (red) from an arterial myocyte.