Heteromultimeric CLC chloride channels with novel properties

C Lorenz

M Pusch

T Jentsch

Center for Molecular Neurobiology, Hamburg University, Martinistrasse 52, D-20246 Hamburg, Germany

^{Present address: BASF AG, ZHA-A30, 67056 Ludwigshafen, Germany.}

^{To whom reprint requests should be addressed.}

Gerhard Giebisch, Yale University, New Haven, CT

Received 1996 Jun 10; Accepted 1996 Sep 3.

Abstract

The skeletal muscle chloride channel CLC-1 and the ubiquitous volume-activated chloride channel CLC-2 belong to a large gene family whose members often show overlapping expression patterns. CLC-1 and CLC-2 are coexpressed in skeletal and smooth muscle and in the heart. By coexpressing CLC-1 and CLC-2 in Xenopus oocytes, we now show the formation of novel CLC-1/CLC-2 heterooligomers that yield time-independent linear chloride currents with a chloride → bromide → iodide selectivity sequence. Formation of heterooligomeric CLC channels increases the number and possible functions of chloride channels.

Keywords: anion channel, multimers, myotonia, cystic fibrosis, cell volume regulation

Abstract

CLC chloride channels form an expanding new gene family with at least nine mammalian members known to date (for review, see refs. 1 and 2). In mammals, they play important roles in cell volume regulation (3), control of muscle excitability (4), and possibly transepithelial transport (5, 6). Two human diseases are known to be caused by defective CLC genes: mutations in the muscle channel CLC-1 can cause recessive (Becker) as well as dominant (Thomsen) myotonia (7, 8), whereas X-linked hereditary hypercalciuric nephrolithiasis is due to mutations in CLC-5 (9, 10).

CLC proteins are structurally unrelated to other known ion channels and span the membrane about 12 times (3, 6, 11, 12). First insights into their structure-function relationship have been obtained for the mechanism of swelling activation of CLC-2 (3). The gating mechanism of the Torpedo channel CLC-0 was studied in detail. It was proposed that the channel is intrinsically voltage-independent, being gated by the permeant anion (12). Finally, analysis of dominant negative mutations found in human myotonia (Thomsen disease) suggested that CLC-1 chloride channels function as homomultimers with probably more than two subunits (8, 13).

Several CLC genes are rather broadly expressed. CLC-2, CLC-6, and CLC-7 are present in every cell and tissue examined (14, 15), while CLC-3 and CLC-4 are expressed in brain, heart, kidney, and, depending on the species, several other tissues (1, 16, 17) Thus, many cells will express several members of the CLC gene family in parallel. Since CLC-1 is known to function as a (homo)multimer (8), this raises the possibility that coexpression of different CLC members in the same cell may lead to the formation of functional heterooligomeric channels with novel properties. Such a situation is, for example, found with potassium channels (18–20) or glutamate receptors (21).

We used coexpression of the easily expressible CLC-1 and CLC-2 chloride channels to test for the formation of heteromultimeric CLC channels. This is also of physiological importance, as both channel mRNAs are coexpressed in several tissues (14, 22).

CLC-1 is very predominantly expressed in skeletal muscle, but low transcript levels could also be detected in kidney, heart, and smooth muscle (22). CLC-1 mediates the majority of muscle membrane chloride conductance, which is unusually high in that tissue (≈70–80% of total resting conductance; ref. 23).

In contrast to CLC-1, CLC-2 is ubiquitously expressed. It is normally closed but can be slowly activated by strong hyperpolarization (14). Another activation mechanism is cell swelling, suggesting a role of CLC-2 in regulatory volume decrease (3). Its expression in apical membranes of airway epithelia renders it interesting for cystic fibrosis (24).

We now show that coexpression of CLC-1 and CLC-2 indeed results in the formation of novel heteromultimeric channels. This demonstrates for the first time that subunits encoded by different CLC genes can assemble to form functional heteromeric channels, and thus adds a further level of complexity to the CLC chloride channel family.

Acknowledgments

This work was supported, in part, by the Deutsche Forschungsgemeinschaft, the Fonds der Chemischen Industrie, and the U.S. Muscular Dystrophy Association.

Acknowledgments

Footnotes

Abbreviations: 9-AC, 9-anthracene carboxylic acid; I–V, current–voltage.

Footnotes

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