Journal: PLoS ONE

Article Title: Regulation of hERG and hEAG Channels by Src and by SHP-1 Tyrosine Phosphatase via an ITIM Region in the Cyclic Nucleotide Binding Domain

doi: 10.1371/journal.pone.0090024

Figure Lengend Snippet: A. i. Representative current traces in response to the voltage-clamp protocol shown in the inset. From the holding potential of −80 mV, 3-s long depolarizing steps were applied between −70 and +60 mV (10-mV increments). This opens the channels, but at positive potentials, some inactivation is seen (c→o→i). The voltage was then stepped to −40 mV to remove inactivation (i→o) and monitor outward tail currents. ii. The current amplitude at the end of each 3-sec long depolarizing step (open arrow in Ai) was used to construct a current-versus-voltage (I–V curve) before and after perfusing the hERG blocker, 1 µM E-4031, into the bath. iii. Summary of instantaneous I–V relations from tail currents (closed arrow in Ai) before and after adding E-4031. In all graphs, the values are mean±SEM for the number of cells in parentheses. B. Representative whole-cell currents are shown before and 25 min after adding 10 µM PP1 to the bath. From a holding potential of −80 mV, a 1 s long pulse to +60 mV was used to activate and then inactivate the channels (c→o→i). Then, inactivation was rapidly removed by a 16 ms long pulse to −120 mV (i→o). Instantaneous tail currents (arrow) during test pulses between −110 and +20 mV (10-mV increments) were used to measure the open-channel current- vs -voltage (I–V) relationship (summarized in D) and inactivation kinetics (summarized in E). C. PP1 reduces the hERG current. To monitor time-dependent changes, the instantaneous tail current at +20 mV was repeatedly measured in control cells and in separate cells exposed to 10 µM PP1. Each current was normalized to its initial value (measured at 3–4 min after starting the recording). Significant differences between control and PP1-treated cells are indicated as * p <0.05; ** p <0.01. D. Open-channel I–V relations from instantaneous tail currents (as in panel B) were recorded at 25 min in control cells and in separate cells exposed to 10 µM PP1. For each cell, the current was normalized to cell capacitance to yield current density (pA/pF). E. The time constant of inactivation (τ) was measured after 25 min in control cells or with 10 µM PP1 in the bath. That is, for test pulses between +20 and −20 mV, the decay of the tail current (o→i; as in panel B) was fitted to a mono-exponential function: I t = AMP *exp(− t/τ ), where I t is the outward current at time t and AMP is the initial current amplitude. Each fit was begun at the time indicated by the arrow in panel B.

Article Snippet: The hERG blocker, E-4031 (Alomone, Jerusalem, Israel), and the hEAG1 blocker, astemizole (Sigma, Oakville, ON, Canada), were used to confirm that the observed current was hERG or hEAG1, respectively.

Techniques: Construct