Potassium channels in the basolateral membrane of the rectal gland of the dogfish (Squalus acanthias)

Abstract

Previous studies in isolated, in vitro perfused rectal gland tubules (RGT) have revealed that the basolateral membrane possesses a K⁺ conductive pathway. In the present study, we have utilized the patch clamp technique in RGT segments to characterize this pathway. The basolateral membrane was approached with patch pipettes at the open end of in vitro perfused segments [5]. Recordings were obtained in cell-attached as well as in excised inside-out patches. In cell-attached patches with the pipette filled with a KCl solution (274 mmol/l) and the bath containing NaCl shark Ringer (275 mmol/l), inward K⁺ currents (from pipette into cell) with a mean slope conductance of 123±26 pS (n=3) were observed. We were unable to generate outward K⁺ currents at high depolarizing (cell more positive) clamp voltages. This indicates inward rectification of this channel. To examine the rectification properties further, excised (inside out) patches were exposed to K⁺ concentration gradients, directed out of, as well as into the pipette. With NaCl in the pipette and KCl in the bath, K⁺ outward currents were observed. The current-voltage (IV) relation revealed Goldman-type rectification, with a mean single channel conductance of 185±28 pS (n=7) at high positive voltages (linear range of the IV curve). The single-channel permeability coefficient for K⁺ was 0.26±0.04 ·10⁻¹² cm³/s (n=7). In the reversed experiment (pipette KCl, bath NaCl), inward currents of similar kinetics and amplitude were obtained. The single channel conductance was 146±21 pS (n=7) at high negative voltages (linear range of the IV curve). The single channel permeability coefficient for K⁺ was 0.21±0.03·10⁻¹² cm³/s (n=7). We were not able to reverse the currents in any of these experiments, indicating that this channel is highly selective for K⁺ over Na⁺. In all three series of experiments, the kinetic appearance of the channels was similar. Bursts of activity were followed by interburst pauses. The open state was described by a single time constant of 3.0±0.2 ms, whereas the closed state was described by two time constants of 0.7±0.2 ms and 2.8±0.5 ms (n=8). It can be concluded that these channels permit K⁺ inward and outward currents. They are probably the equivalent of the basolateral K⁺ conductance as observed in a previous study [12]. Under physiological conditions a single channel conductance of some 20 pS is predicted from the present data. In cell-attached patches, with a high K⁺ concentration in the pipette, the channel behaves as an inward rectifier.