SIRK is an inward-rectifying voltage-gated
channel, as assessed by macroscopic current analysis in X.
laevis oocytes. A through C, Analysis by two-electrode
voltage-clamp. The bath solution contained 1 mm
CaCl2, 1.5 mm
MgCl2, 5 mm HEPES
[4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid]-NaOH (pH 7.4),
and 100 mm (K + Na) Cl (K+
concentration indicated below). A, SIRK currents elicited by 1.5-s
voltage pulses from 0 to −165 mV in 12 steps (−15-mV increments) from
a holding potential of −40 mV in a 100 mm
K+ solution. B, The steady-state current at the
end of the activation step was plotted against membrane potential for
three different external K+ concentrations (in
mm): 100 (circles), 10 (squares), and 2
(triangles). Inset, Plotting the reversal potential
(Erev) for the SIRK current (black circles)
versus the external concentration of K+ revealed
that Erev shifted by 56.5 mV for a 10-fold
increase in external K+ concentration, as
expected for a highly selective K+ channel. Solid
line in the inset, K+ equilibrium potential
(EK) calculated with the Nernst equation
assuming a 113 mm intracellular
K+ concentration (i.e.
Erev = EK in
100 mm external K+). C,
SIRK activation level at steady state for three different K
concentrations (in mm): 100 (diamonds), 10
(squares), and 2 (triangles); n = 3 oocytes. A
two-state Boltzmann relation (dotted line; Lacombe and Thibaud, 1998)
modeled the G/Gmax ratio. D, Analysis of
macroscopic cur- rents assessed by patch-clamp (patch-excision and
patch-cramming). Both bath and pipette solutions contained 100
mm KCl, 2 mm
MgCl2, and 10 mm HEPES-NaOH
(pH 7.4). In the cell-attached patch clamp configuration, the
current-voltage curve (1) is reminiscent of those obtained by
two-electrode voltage-clamp (see B). After patch excision (inside-out
configuration), the current amplitude decreased very quickly (trace 2,
obtained 10 s after the patch excision). Patch cramming into the
oocyte produced an increase in current (trace 3). Data shown are
representative of five independent patch-cramming experiments.