Assembly and Suppression of Endogenous Kvl.3 Channels in Human T Cells

The predominant K + channel in human T lymphocytes is Kvl.3, which inactivates by a C-type mechanism. To study assembly of these tetrameric channels in Jurkat, a human T-lymphocyte cell line, we have characterized the formation of heterotetrameric channels between endogenous wild-type (WT) Kvl.3 subunits and heterologously expressed mutant (A413V) Kvl.3 subunits. We use a kinetic analysis of C-type inactivation of currents produced by homotetrameric channels and heterotetrameric channels to determine the distribution of channels with different subunit stoichiometries. The distributions are welldescribed by either a binomial distribution or a binomial distribution plus a fraction of WT homotetramers, indicating that subunit assembly is a random process and that tetramers expressed in the plasma membrane do not dissociate and reassemble. Additionally, endogenous Kvl.3 current is suppressed by a heterologously expressed truncated Kvl.3 that contains the amino terminus and the first two transmembrane segments. The time course for suppression, which is maximal at 48 h after transfection, overlaps with the time interval for heterotetramer formation between heterologously expressed A413V and endogenous WT channels. Our findings suggest that diversity o fK + channel subtypes in a cell is regulated not by spatial segregation of monomeric pools, but rather by the degree of temporal overlap and the kinetics of subunit expression. I N T R O D U C T I O N H u m a n T lymphocytes contain voltage-gated K + channels (400-750 channels /ce l l [Cahalan et al., 1985; Deutsch et al., 1986; Deutsch et al., 1991]). These channels, composed of four identical subunits (MacKinnon, 1991), are encoded by the Kvl.3 gene (Cai et al., 1992) and de te rmine the resting m e m b r a n e potential in human T cells (Leonard et al., 1992). At its resting potential of 7 0 mV (Deutsch et al., 1979; Rink et al., 1980; Gelfand et al., 1984), the T cell has an extremely high input impedance, ~ 2 0 Gohms, and thus opening of only one K + channel is sufficient to drive the membrane toward more negative m e m b r a n e potentials (Lee et al., 1992). At least two impor tan t functions in the T cell depend on m e m b r a n e potential. First, volume regulation in response to hypotonic shock is de te rmined by electrochemical driving forces (Grinstein and Smith, 1990; Deutsch and Chen, 1993), and second, mitogenic interleukin-2 product ion and proliferation is sensitive to m e m b r a n e potential, thus implicating a role for Address correspondence to Dr. Carol Deutsch, Department of Physiology, University of Pennsylvania, 3700 Hamilton Walk, Richards Bldg, D-203, Philadelphia, PA 19104-6085. Kvl.3 in mitogenesis (Bono et al., 1989; Freedman et al., 1992; Lin et al., 1993). There fore it is impor tan t to unders tand what governs not only the biophysical propert ies of these channels but also the format ion of functional K + channels. The density of K + channels at the surface of the cell depends on transcriptional and translational regulation of subunits, their association to form tetramers, and the integrat ion of these tetramers into the plasma membrane . How and where Kvl.3 subunits assemble is not known, nor whether these tetrameric channels in the plasma m e m b r a n e are in equil ibrium with m o n o m e r i c pools of subunits. For most plasma m e m b r a n e proteins mult imerizat ion occurs posttranslationally in the endoplasmic ret iculum (ER) and is often a prerequisite for efficient expor t of these proteins f rom the ER (Doms et al., 1993; Hurt ley and Helenius, 1989). Proper folding of protein subunits produces the required topology for specific quaternary contacts allowing mult imerizat ion between appropr ia te assembly partners. The time for assembly of different proteins ranges f rom minutes to hours after synthesis (Carlin and Merlie, 1986). Although the ER is the primary site of multimerization, o ther compar tments later in the secretory pathway can host fur ther assembly reactions (Rotundo, 1984; Sporn et al., 1986). 409 J. GEN. PHYSIOL. 9 The Rockefeller University Press ~ 0022-1295/96/03/409/12 $2.00 Volume 107 March 1996 409-420 on F ebuary 3, 2013 jgp.rress.org D ow nladed fom Published March 1, 1996