An open rectifier potassium channel with two pore domains in tandem cloned from rat cerebellum

Abstract

Tandem pore domain K+ channels represent a new family of ion channels involved in the control of background membrane conductances. We report the structural and functional properties of a TWIK-related acid-sensitive K+ channel (rTASK), a new member of this family cloned from rat cerebellum. The salient features of the primary amino acid sequence include four putative transmembrane domains and, unlike other cloned tandem pore domain channels, a PDZ (postsynaptic density protein, disk-large, zo-1) binding sequence at the C terminal. rTASK has distant overall homology to a putative Caenorhabditis elegans K+ channel and to the mammalian clones TREK-1 and TWIK-1. rTASK expression is most abundant in rat heart, lung, and brain. When exogenously expressed in Xenopus oocytes, rTASK currents activate instantaneously, are noninactivating, and are not gated by voltage. Because rTASK currents satisfy the Goldman-Hodgkin-Katz current equation for an open channel, rTASK can be classified an open rectifier. Activation of protein kinase A produces inhibition of rTASK, whereas activation of protein kinase C has no effect. rTASK currents were inhibited by extracellular acidity. rTASK currents also were inhibited by Zn2+ (IC50 = 175 microM), the local anesthetic bupivacaine (IC50 = 68 microM), and the anti-convulsant phenytoin ( approximately 50% inhibition at 200 microM). By demonstrating open rectification and open probability independent of voltage, we have established that rTASK is a baseline potassium channel.

Fig. 1.

Sequence analysis of rTASK. A, Nucleotide and deduced amino acid sequence of rTASK. The four putative transmembrane domains (M1–M4) are enclosed inboxes. Underlined segments indicate pore regions (P1, P2). Sites for N-linked glycosylation (asterisk) and phosphorylation by tyrosine kinase (filled circle), protein kinase C (filled squares), and protein kinase A (filled triangles) are indicated. Thecircled amino acids at the C terminal indicate the postsynaptic density (PSD) binding motif. B, Hydropathy plot showing transmembrane domains (M1–M4) and the P regions (P1, P2) using the Kyte–Doolittle algorithm. C, Predicted transmembrane topology of rTASK with labeled transmembrane domains and pore regions. The GenBank accession number of the rTASK clone is AF031384.

Fig. 2.

Sequence comparison of related tandem pore domains. Protein sequence alignments (dark areas) of the P1, post-P1, P2, and post-P2 regions of rTASK with the homologous regions of the three most closely related C. eleganstandem pore domain K⁺ channels and of TREK-1, TWIK-1, ORK1, and TOK1 are shown.

Fig. 3.

Northern blot analysis of rTASK distribution in adult rat tissue. A rat multiple tissue Northern blot was probed at high stringency with a probe made from the EST400 sequence. The blot was reprobed with a β-actin cDNA probe for a control. Added lane shows the presence of rTASK transcript in rat cerebellum.

Fig. 4.

Biophysical properties of rTASK currents inXenopus oocytes studied with two-electrode voltage clamp. A, Reversal potential as a function of extracellular K⁺ for rTASK-expressing oocytes. Reversal potential changed by 54 ± 3 mV per 10-fold change in extracellular K⁺, as estimated with linear regression (regression line shown). B, Whole-cell current–voltage relation with either 5 mm(filled circles) or 100 mm(open circles) extracellular K⁺. The current–voltage relations for an open K⁺-selective channel estimated from the Goldman–Hodgkin–Katz current equation are drawn as solid lines.

Fig. 5.

Extracellular pH sensitivity of rTASK.A, Representative current responses from rTASK cRNA-injected oocytes at pH 7.6 and 6.4 (voltage pulses from −120 to +40 mV). B, Current–voltage curves of rTASK-injected oocytes at several different extracellular pH values. Currents from control saline-injected oocytes were unchanged over this pH range.C, Effect of extracellular pH on rTASK currents (−80 to +40 mV pulse). Data have been normalized to currents measured at pH 7.6. Mean values are shown with the SE.

Fig. 6.

Concentration–response curve for bupivacaine. Currents elicited by the −80 to +40 mV pulse have been normalized to currents measured before and after bupivacaine application and fit to a logistic function (IC₅₀ = 68 μm; Hill coefficient = 0.6).

Fig. 7.

Comparative pharmacology of tandem pore domain K⁺ channels expressed in Xenopusoocytes. Relative responses of three clones (rTASK, ORK1, and TOK1) are compared for several potent modulators (extracellular pH 6.4, bupivacaine 1 mm, lidocaine 1 mm, and Zn²⁺ 100 μm). Studies were performed under two-electrode voltage clamp in frog Ringer’s solution at pH 7.6. Response is defined as the current measured for the −80 to +40 mV pulse during the treatment condition compared with control. Mean values are shown with SE. Numbers over the barsindicate number of experiments.

Fig. 8.

Patch-clamp recordings of rTASK currents expressed in Xenopus oocytes. A, Unitary rTASK currents recorded from an outside-out patch at several holding potentials. The recording pipette was filled with 150 mmK-aspartate, and the external solution was 150 mm NaCl. Currents were filtered at 2 kHz. B, Compressed records of single rTASK channel activity.

Fig. 9.

rTASK single-channel properties recorded from outside-out patches. A, Single-channelI–V relations. The recordings were made with 150 mm K⁺ in the recording pipette and bath solutions of 150 mm NaCl (circles;n = 7), a mixture of 75 mmNa⁺ with 75 mm K⁺(triangles; n = 2), and 150 mm K⁺ (squares;n = 4). In the presence of symmetrical 150 mm K⁺, the I–V relation was best fit to a linear function. Data in the other conditions were fit with third degree polynomial functions, which illustrates the pattern of outward rectification. The unitary current was measured as the amplitude of the current from the closed channel level to a cursor positioned in the center of the open channel noise. Error bars indicate SD of the mean. B, Independence of the open probability of single rTASK channels from the patch potential. The open probabilities are the means from outside-out patches (n = 4) recorded with a bath solution of 150 mm NaCl. Individual values were calculated by setting the single-channel amplitude to unity and integrating records from 30 sec data segments at each voltage. Error bars indicate SDs.