Structure of the KvAP voltage-dependent K ⁺ channel and its dependence on the lipid membrane

Abstract

Voltage-dependent ion channels gate open in response to changes in cell membrane voltage. This form of gating permits the propagation of action potentials. We present two structures of the voltage-dependent K ؉ channel KvAP, in complex with monoclonal Fv fragments (3.9 Å) and without antibody fragments (8 Å). We also studied KvAP with disulfide cross-bridges in lipid membranes. Analyzing these data in the context of the crystal structure of Kv1.2 and EPR data on KvAP we reach the following conclusions: (i) KvAP is similar in structure to Kv1.2 with a very modest difference in the orientation of its voltage sensor; (ii) mAb fragments are not the source of non-native conformations of KvAP in crystal structures; (iii) because KvAP contains separate loosely adherent domains, a lipid membrane is required to maintain their correct relative orientations, and (iv) the model of KvAP is consistent with the proposal of voltage sensing through the movement of an argininecontaining helix-turn-helix element at the protein-lipid interface.membrane protein ͉ protein-lipid interface ͉ voltage-gated ion channel ͉ voltage sensor V oltage-dependent ion channels ''sense'' voltage differences across the cell membrane and open or close in response to its value (1). These channels contain a centrally located pore surrounded by four voltage sensors. Voltage-dependent K ϩ (Kv) channels are tetramers with four identical subunits, each with six transmembrane segments (S1-S6): S5 and S6 form the central pore at the interface between the subunits, and S1-S4 form the voltage sensors (2, 3). The voltage sensors have four to seven positively charged amino acids (usually arginine) on S4, known as gating charges, and fewer negatively charged amino acids (aspartate or glutamate) distributed on S1, S2, and S3. The voltage sensors undergo a conformational change when the pore gates open, coupling movement of the S4 gating charges within the membrane electric field to channel open probability.The first structure of a Kv channel, termed KvAP, from the archeabacterium Aeropyrum Pernix (4), was determined by crystallizing the channel as a complex with a monoclonal Fab fragment attached to its voltage sensors [Protein Data Bank (PDB) ID code 1ORQ] (5). In that structure the voltage sensors are in a non-native conformation, displaced toward the intracellular side of the transmembrane pore. Together with a second structure of the voltage sensor alone, termed the isolated voltage sensor (PDB ID code 1ORS), the following ideas about voltagedependent gating were proposed (5, 6): that the voltage sensor is a very mobile structure, presumably because it must carry charged amino acids through the membrane electric field; that the charge bearing S4 forms a helix-turn-helix with the Cterminal half of S3 (S3b), and that the helix-turn-helix moves at the protein-lipid interface a large distance. Experiments using avidin capture of biotin linked to the voltage sensor indicated that four of the S4 arginine amino acids translate 15-20 Å across the membrane. These struc...

Dimeric subunit stoichiometry of the human voltage-dependent proton channel Hv1

“…Forcing conditions also result in weak cross-links in two new regions: the S2-S3 turn, which is also known as the membrane interface anchor (positions 164 and 173) (7), and the tip of the voltage sensor paddle (positions 187 and 194) (Fig. 3 c and d) (6).…”

Portability of paddle motif function and pharmacology in voltage sensors

“…HaTx does not interact tightly with either KvAP or Kv1.2, the two Kv channels for which X-ray structures are available, and the sequence similarity between these channels and Kv2.1 is quite low, precluding assignment of crucial residues within the existing structures. The interaction of VSTx1 with chimeras containing the entire paddle of KvAP (Fig 2), however, offers an opportunity to explore the structure of a tarantula toxin receptor because there are three X-ray structures of KvAP 1,8 , and the structure of the paddle motif in these is very similar (whether or not an antibody is bound). To define critical residues within the paddle motif that may interact with VSTx1, we Ala scanned the paddle region of the C*[S3-S4]AP chimera between KvAP and Shaker.…”

Functional diversity of potassium channel voltage-sensing domains

“…It is now recognized that the first KvAP structure was likely distorted by crystallization conditions and its structure has been corrected and shown to be consistent with later structures of other VSDs. 22,23 Following the structure of KvAP, the next VSD to be defined at atomic level and considered a native, non-perturbed structure was part of the now paradigmatic structure of the Kv1.2 mammalian potassium channel. 24 This structure showed that the VSDs interact with the pore domain of an adjacent subunit and stand at the periphery of the whole channel.…”