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Membrane-embedded voltage-sensor domains in voltage-dependent potassium channels ([K.sub.v] channels) contain an impressive number of charged residues. How can such highly charged protein domains be efficiently inserted into biological membranes? In the plant [K.sub.v] channel KAT1, the S2, S3, and S4 transmembrane helices insert cooperatively, because the S3, S4, and S3-S4 segments do not have any membrane insertion ability by themselves. Here we show that, in the Drosophila Shaker [K.sub.v] channel, which has a more hydrophobic S3 helix than KAT1, S3 can both insert into the membrane by itself and mediate the insertion of the S3-S4 segment in the absence of S2. An engineered KAT1 S3-S4 segment in which the hydrophobicity of S3 was increased or where S3 was replaced by Shaker S3 behaves as Shaker S3-S4. Electrostatic interactions among charged residues in S2, S3, and S4, including the salt bridges between E283 or E293 in S2 and R368 in S4, are required for fully efficient membrane insertion of the Shaker voltage-sensor domain. These results suggest that cooperative insertion of the voltage-sensor transmembrane helices is a property common to [K.sub.v] channels and that the degree of cooperativity depends on a balance between electrostatic and hydrophobic forces. charged residue | [K.sub.v] channel | salt bridge | translocation

KAT1 is a member of the Shaker family of voltage-dependent [K.sup.+] channels, which has six transmembrane segments (called S1-S6), including an amphipathic S4 with several positively charged residues and a hydrophobic pore-forming region (called P) between S5 and S6. In this study, we systematically evaluated the function of individual and combined transmembrane segments of KAT1 to direct the final topology in the endoplasmic reticulum membrane by in vitro translation and translocation experiments. The assay with single-transmembrane constructs showed that S1 possesses the type II signal-anchor function, whereas S2 has the stop-transfer function. The properties fit well with the results derived from combined insertion of S1 and S2. S3 and S4 failed to integrate into the membrane by themselves. The inserted glycosylation sequence at the S3-S4 loop neither prevented the translocation of S3 and S4 nor impaired the function of voltage-dependent [K.sup.+] transport regardless of the changed length of the S3-S4 loop. S3 and S4 are likely to be posttranslationally integrated into the membrane only when somewhat specific interaction occurs between them. S5 had the ability of translocation reinitiation, and S6 had a strong preference for [N.sub.exo]/[C.sub.cyt] orientation. The pore region resided outside because of its lack of its transmembrane-spanning property. According to their own topogenic function, combined constructs of S5-P-S6 conferred the membrane-pore-membrane topology. This finding supports the notion that a set of S5-P-S6 can be independently integrated into the membrane. The results in this study provide the fundamental topogenesis mechanism of transmembrane segments involving voltage sensor and pore region in KAT1.

The Arabidopsis thaliana AtHKT1 protein, a [Na.sup.+]/[K.sup.+] transporter, is capable of mediating inward [Na.sup.+] currents in Xenopus laevis oocytes and [K.sup.+] uptake in Escherichia coli. HKT1 proteins are members of a superfamily of [K.sup.+] transporters. These proteins have been proposed to contain eight transmembrane segments and four pore-forming regions arranged in a mode similar to that of a [K.sup.+] channel tetramer. However, computer analysis of the AtHKT1 sequence identified eleven potential transmembrane segments. We have investigated the membrane topology of AtHKT1 with three different techniques. First, a gene fusion alkaline phosphatase study in E. coli clearly defined the topology of the N-terminal and middle region of AtHKT1, but the model for membrane folding of the C-terminal region had to be refined. Second, with a reticulocyte-lysate supplemented with dog-pancreas microsomes, we demonstrated that N-glycosylation occurs at position 429 of AtHKTI. An engineered unglycosylated protein variant, N429Q, mediated [Na.sup.+] currents in X. laevis oocytes with the same characteristics as the wild-type protein, indicating that N-glycosylation is not essential for the functional expression and membrane targeting of AtHKT1. Five potential glycosylation sites were introduced into the N429Q. Their pattern of glycosylation supported the model based on the E. coli-alkaline phosphatase data. Third, immunocytochemical experiments with FLAG-tagged AtHKT1 in HEK293 cells revealed that the N and C termini of AtHKT1, and the regions containing residues 135-142 and 377-384, face the cytosol, whereas the region of residues 55-62 is exposed to the outside. Taken together, our results show that AtHKT1 contains eight transmembrane-spanning segments.

Membrane-embedded voltage-sensor domains in voltage-dependent potassium channels (Kv channels) contain an impressive number of charged residues. How can such highly charged protein domains be efficiently inserted into biological membranes? In the plant Kv channel KAT1, the S2. S3, and 54 transmembrane helices insert cooperatively, because the S3, 54, and 53-54 segments do not have any membrane insertion ability by themselves. Here we show that, in the Drosophila Shaker Kv channel, which has a more hydrophobic S3 helix than KAT1, 53 can both insert into the membrane by itself and mediate the insertion of the S3-54 segment in the absence of 52. An engineered KAT1 53-54 segment in which the hydrophobicity of 53 was increased or where 53 was replaced by Shaker 53 behaves as Shaker S3-54. Electrostatic interactions among charged residues in 52, S3, and 54, including the salt bridges between E283 or E293 in S2 and R368 in S4, are required for fully efficient membrane insertion of the Shaker voltage-sensor domain. These results suggest that cooperative insertion of the voltage-sensor transmembrane helices is a property common to Kv channels and that the degree of cooperativity depends on a balance between electrostatic and hydrophobic forces. [ABSTRACT FROM AUTHOR]

Describes the integration of Shaker-type K[sup +] channel, KAT1 into the endoplasmic reticulum (ER) membrane. Effect of the inserted glycosylation sequence; Accounts on the topogenesis mechanism transmembrane segments; Accession of oligosaccharyl transferase in the ER membrane.