New Insights on the Voltage Dependence of the K Ca 3 . 1 Channel Block by Internal

We present in this work a structural model of the open IKCa (K Ca 3.1) channel derived by homology modeling from the MthK channel structure, and used this model to compute the transmembrane potential profile along the channel pore. This analysis showed that the selectivity filter and the region extending from the channel inner cavity to the internal medium should respectively account for 81% and 16% of the transmembrane potential difference. We found however that the voltage dependence of the IKCa block by the quaternary ammonium ion TBA applied internally is compatible with an apparent electrical distance of 0.49 0.02 ( n 6) for negative potentials. To reconcile this observation with the electrostatic potential profile predicted for the channel pore, we modeled the IKCa block by TBA assuming that the voltage dependence of the block is governed by both the difference in potential between the channel cavity and the internal medium, and the potential profile along the selectivity filter region through an effect on the filter ion occupancy states. The resulting model predicts that should be voltage dependent, being larger at negative than positive potentials. The model also indicates that raising the internal K concentration should decrease the value of measured at negative potentials independently of the external K concentration, whereas raising the external K concentration should minimally affect for concentrations 50 mM. All these predictions are born out by our current experimental results. Finally, we found that the substitutions V275C and V275A increased the voltage sensitivity of the TBA block, suggesting that TBA could move further into the pore, thus leading to stronger interactions between TBA and the ions in the selectivity filter. Globally, these results support a model whereby the voltage dependence of the TBA block in IKCa is mainly governed by the voltage dependence of the ion occupancy states of the selectivity filter. key words: calcium-activated potassium channel • quaternary ammonium • single file diffusion • EBIO • modeling I N T R O D U C T I O N Ca 2 -activated potassium channels (K(Ca 2 )) are present in most mammalian cell types, where their primary role is to establish a link between the various Ca 2 -based second messenger systems and the electrical properties of the cells. Three main classes of Ca 2 -activated potassium channels have been to date identified on the basis of their permeation properties and pharmacology (Vergara et al., 1998). These include the charybdotoxinand iberiotoxin-sensitive K Ca 1.1 channels of large conductance (150–220 pS); the intermediate conductance (20–50 pS) IKCa channels (K Ca 3.1) inhibited by clotrimazole (Rittenhouse et al., 1997) and TRAM34 (Wulff et al., 2001), and the apamine-sensitive and -insensitive SK channels of small conductance (K Ca 2.1, K Ca 2.2, K Ca 2.3) (Kohler et al., 1996). In contrast to K Ca 1.1, the SK and IKCa channel gating process is voltage insensitive, and the Ca 2 sensitivity in both channels is conferred by the Ca 2 -binding protein, calmodulin, constitutively bound to the channel proximal COOH-terminal regions (Khanna et al., 1999). An important contribution to our understanding of the IKCa channel molecular identity came from the cloning of the hKCa4, hIK1, and hSK4 channels (Ishii et al., 1997; Joiner et al., 1997; Logsdon et al., 1997; Vandorpe et al., 1998; Warth et al., 1999). The IKCa channel is a tetrameric protein with each subunit comprising 427 amino acids organized in six transmembrane segments TM1–TM6 with a pore motif between segments 5 and 6. The three-dimensional (3D) structure of IKCa, however, remains unresolved. Docking simulations and binding studies using charybdotoxin analogues have revealed that the IKCa external vestibule is structurally similar to the external pore region of Address correspondence to Rémy Sauvé, Département de Physiologie, Membrane Protein Study Group, Université de Montréal, C.P. 6128, Succursale Centre-ville, Montréal, Québec H3C 3J7, Canada. Fax: (514) 343-7146. email: [email protected] Abbreviations used in this paper: EBIO, 1-ethyl-2-benzimidazolinone; MTS, methanethiosulfonate; MTSET, [2-(trimethylammonium) ethyl]methanethiosulfonate bromide; NMDG, N -methyld -glucamine; QA, quaternary ammonium; SCAM, substituted cysteine accessibility method; TBA, tetrabutylammonium. on Jne 1, 2017 D ow nladed fom Published September 27, 2004