Abstract
Kv1.1 and Kv1.4 potassium channels are expressed as mature glycosylated proteins in brain, whereas they exhibited striking differences in degree of trans-Golgi glycosylation conversion and high cell surface expression when they were transiently expressed as homomers in cell lines. Kv1.4 exhibited a 70% trans-Golgi glycosylation conversion, whereas Kv1.1 showed none, and Kv1.4 exhibited a approximately 20-fold higher cell surface expression level as compared with Kv1.1. Chimeras between Kv1.4 and Kv1.1 and site-directed mutants were constructed to identify amino acid determinants that affected these processes. Truncating the cytoplasmic C terminus of Kv1.4 inhibited its trans-Golgi glycosylation and high cell surface expression (as shown by Li, D., Takimoto, K., and Levitan, E. S. (2000) J. Biol. Chem. 275, 11597-11602), whereas truncating this region on Kv1.1 did not affect either of these events, indicating that its C terminus is not a negative determinant for these processes. Exchanging the C terminus between these channels showed that there are other regions of the protein that exert a positive or negative effect on these processes. Chimeric constructs between Kv1.4 and Kv1.1 identified their outer pore regions as major positive and negative determinants, respectively, for both trans-Golgi glycosylation and cell surface expression. Site-directed mutagenesis identified a number of amino acids in the pore region that are involved in these processes. These data suggest that there are multiple positive and negative determinants on both Kv1.4 and Kv1.1 that affect channel folding, trans-Golgi glycosylation conversion, and cell surface expression.
Highlights
Potassium (Kϩ) channels are expressed by most cells and play important roles in cell physiology, including setting the resting membrane potential and repolarizing/modulating action potential waveforms in excitable tissues [1,2,3]
Because Kv1.4 has a smaller single channel conductance (Kv1.4 ϭ 4 picosiemens and Kv1.1 ϭ 10 picosiemens), fast N-type inactivation, and fast C-type inactivation compared with Kv1.1 [6], we predict that the ϳ10-fold higher difference for Kv1.4 versus Kv1.1 recorded in Fig. 5B was more likely closer to ϳ20-fold difference in surface protein expression, and biotinylation of surface glycoproteins suggests that this is the case (Fig. 5, C and D)
Kv1.4 and Kv1.1 exhibited very different degrees of efficient trans-Golgi glycosylation and high cell surface expression when expressed as homomers in cell lines, and for both processes, Kv1.4 showed a higher level of glycosylation conversion and surface expression as compared with Kv1.1
Summary
Potassium (Kϩ) channels are expressed by most cells and play important roles in cell physiology, including setting the resting membrane potential and repolarizing/modulating action potential waveforms in excitable tissues [1,2,3]. Functional channels appear to be composed of noncovalently associated homo- and/or heteromeric tetramers of subunits within a subfamily, and numerous amino acid determinants involved in channel operation have been identified [4, 5]. Endo H cleaves only high mannose-type immature N-linkages found on ER1 glycoproteins and some plasma membrane proteins, whereas PNGase F cleaves all N-linkages (high mannose-, hybrid-, and complex-type linkages) [14] These findings imply that these glycoproteins were efficiently processed and sialidated in the trans-Golgi in native brain tissue. Determinants Involved in Kϩ Channel Subunit Processing tants to map amino acid determinants involved in their proper folding in the ER, efficient trans-Golgi glycosylation conversion, and high cell surface expression
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