Table 1.
PCR primers for generating Slick/Slick, Slack/Slack and Slick/Slack concatemeric channels.
Fig 1.
Volume regulation of Slick and Slack homomeric and heteromeric channels.
Co-expression of Slick, Slack and AQP1 in Xenopus laevis oocytes. Currents were stimulated by a pulse protocol (inset in A). Representative currents at + 80 mV (A, C, E), as well as maximal currents normalized to isotonic buffers (B, D, F) are shown for oocytes exposed from isotonic (black) to hypotonic (light grey) and hypertonic buffers (dark grey). (A, B) Slick+AQP1. (C, D) Slack+AQP1. (E, F) Slick+Slack+AQP1. (G) Current–Voltage relationship for oocytes expressing homomeric Slick or Slack channels and oocytes co-expressing both subunits together with AQP1. Currents were stimulated by a step protocol (inset) and were measured at the end of the depolarizing steps. (H) Normalized current-voltage relationship for Slick+AQP1, Slack+AQP1 and Slick+Slack+AQP1 (n = 4–10).
Fig 2.
Slick/Slack concatemeric channels.
A Slick/Slack concatemeric subunit was constructed by the junction of the C-terminus of the Slick channel sequence with the N-terminus of Slack as represented in (A) and expressed in Xenopus laevis oocytes. (B) Representative heteromeric Slick/Slack currents recorded during osmotic challenges, upon stimulation by a pulse protocol as in Fig 1. (C) Summarized data of the effect of osmotic challenges on heteromeric channels. (D). I/V curves normalized to the maximal current for oocytes expressing the Slick/Slack concatemer or co-expressing homomeric Slick and Slack together with AQP1 (n = 10).
Fig 3.
Volume sensitivity of homomeric and heteromeric Slick/Slack channels in different configurations.
In (A), Slack homodimers were synthesized and expressed in Xenopus laevis oocytes. (B) Maximal currents at the end of a depolarizing step to +80 mV for a representative oocyte expressing Slack/Slack, during cell volume challenges. Currents were stimulated by a pulse protocol as in Fig 1. (C) Representative maximal currents over time for a Slack/Slack expressing oocyte exposed to osmotic challenges. The time and period of application of hypotonic and hypertonic buffers are indicated by white and black boxes, respectively. Currents were normalized to isotonic conditions. (D) Maximal currents at the end of the +80 mV step for Slack/Slack expressing oocytes upon volume changes (n = 17). Currents are shown relative to isotonic buffers. (E) Heteromeric Slick/Slack channels formed by 3 Slack and 1 Slick subunits and the volume sensitivity of a representative oocyte (F, G) and summarized data (n = 6) (H). In (I), heteromeric channels were formed by 2 Slick and 2 Slack subunits and their sensitivity to cell volume is represented in (J), (K) and (L) (n = 6). (M) Heteromeric channels made by 3 Slick and 1 Slack subunit and their response osmotic challenges for a representative recording in (N, O), and for 10 oocytes in (P). In (Q), Slick homodimers were constructed and expressed in oocytes. (R) and (S) show current changes upon changes in cell volume for representative oocytes, or for 9 oocytes (T).
Fig 4.
Swelling and shrinking response of homomeric and heteromeric Slick/Slack channels.
Maximal currents measured at the end of +80 mV and normalized to isotonic buffers for homomeric Slack, and Slick, as well as heteromeric Slick/Slack in different configurations (Slick 1:3 Slack; Slick 2:2 Slack; Slick 3:1 Slack) in hypotonic (A) and hypertonic (B) buffers (n = 6–10)