Identification of the S1-S2 linker as a novel mechanosensor for Kv1.5 regulation

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Background: Voltage-gated K+ (Kv) channels are pore-containing membrane proteins. They can be open or closed depending on membrane voltage. When open, they selectively allow K+ to flow across the membrane, generating electrical currents that are critical for the function of all excitable tissues such as the brain, the heart, muscles, and endocrine glands. Kv channels are formed by 4, often identical, subunits. Each subunit contains 6 transmembrane segments, S1 to S6, and intracellular N- and C-termini. While the opening and closing (gating) of Kv channels are controlled by membrane voltages, accumulating evidence indicates that they are also regulated by mechanical force exerted on the cell membrane. However, to date, a structural component of the channel that directly links mechanical force and Kv channel activity has not been described. Hypothesis: The extracellularly-localized S1-S2 linker of Kv1.5 represents a novel mechanosensor for Kv1.5 regulation. Deformation of the S1-S2 linker induced by mechanical force can induce a conformational change of the intracellularly-localized N-terminus of the channel, leading to an altered N-terminus-Src tyrosine kinase interaction, and consequently changed channel activity. Methods: We will construct wild type and mutant Kv1.5 cDNAs in the pcDNA3 vector. We will transfect HEK cells with the cDNA constructs. We will use biochemical techniques to determine protein expression, and use the patch clamp method to record Kv1.5 channel current. We will increase cell volume, and thus membrane tension, by lowering the osmolarity of cell culture medium from 315 to 211 mOsm/L (low osmolarity, LO) and examine the effects of LO on wild type and mutant Kv1.5 channels. Objective 1. Identify that the S1-S2 linker of Kv1.5 is a mechanosensor. Compared to other K+ channels, the S1-S2 linker of Kv1.5 is unusually long (54 versus 5-27 amino acid residues) with many (12) extra proline residues. We will determine whether LO selectively increases Kv1.5 current (compared with other Kv channels such as Kv1.1 and Kv4.3).  Objective 2. Identify that a deformation of S1-S2 linker by LO can impact the conformation of N-terminus of Kv1.5 channels. Objective 3. Identify that the S1-S2 linker regulates Kv1.5 function through altering the interaction between the N-terminus and Src kinases. Src tyrosine kinase inhibits Kv1.5 current through targeting the motif 65-RPLPPLPDPGVRPLPPLP-82. We propose Kv1.5 is constitutively inhibited by endogenous Src tyrosine kinase. We will disrupt Src-Kv1.5 interaction by using Src inhibitors or by mutating the Src binding motif, and examine the effects of LO on Kv1.5 current. Significance: This study is the first to identify a mechanosenser within a Kv channel, Kv1.5, which is fundamental to understanding channel function and discovering novel means of channel regulation. By implementing this hypothesis-driven proposal, HQP will be trained in cutting-edge technologies and critical-thinking skills.