The Voltage-dependent Property Of The SLC26A Family

The auditory system of mammals has high sensitivity and accuracy. This is because cochlear will amplify the sound signals in the inner ear. Amplifier of mammals arise based on outer hair cells (OHCs) somatic electromotility. OHCs of the mammal can change length when give it stimulated electrically. It is named electromotility. The molecular motor of the electromotility is prestin which is the constituent membrane protein of the OHCs. The conformational change of prestin which uses bound C1-ions as voltage sensors to detect the membrane potential results in somatic motility of OHCs. We use nonlinear capacitance (NLC) to represent electromotility.Prestin’s amino acid sequence shows that it belongs to an anion-transporter family, solute carrier protein26A (SLC26A) family and prestin is the fifth member of it. The SLC26A transporter family consists of11members (A1-A11). They are expressed in different tissues and targeted to the membrane of epithelial cells. They share the similar structure, including transmembrane domains, as well as cytoplasmic N-and C-termini. The similar structure suggests the similar function. In mammals, individual members of this family serve two fundamentally distinct functions, transport anion and motor fuction which is the unique property of prestin (A5). However, prestin (A5) has transport property as others in non-mammalian vertebrates. So this observation suggests that motor function of prestin (A5) is the selective production during the evolution. Prestin (A5) is a voltage-dependent motor protein in mammals and voltage-dependent property is very important for its motor function because prestin (A5) need to detect the membrane potential first and then change its length. To determine whether the voltage-dependent property is the common feature of the SLC26A family or the unique property of prestin (A5), we need to study on the voltage-dependent property of other members except A5(prestin).In our study, SLC26A protein sequences from NCBI were used for generating an unrooted phylogenetic tree and we choosed one member from each branch (A2, A4, A6, A7, A9, A11) to study. We measured the membrane capacitance with whole cell voltage-clamp techniques of human embryonic kidney293cells (HEK-293) which expressed DNA of the SLC26A family in order to know the voltage-dependent property of the SLC26A family. Firstly, full-length cDNAs of the SLC26A family were cloned into the pEGFP-N1vectors, respectively, to generate enhanced green fluorescent protein (EGFP) fusion proteins. Then, through plasmid transformation and plasmid extraction steps, DNAs used for study were produced. Secondly, the DNA constructs were introduced into the HEK-293cells using lipofectamine2000. Thirdly, membrane capacitance was measured using whole cell voltage-clamp techniques. We gived a two-sine-wave voltage stimulus protocol (10-mV peak at both390.6and781.2Hz) to cells with robust membrane-associated EGFP expression under fluorescent illumination for membrane capacitance measurements. We compared capacitance measurements of SLC26A family members to get its voltage-dependent property. Intracellular and extracellular solution were changed for studing the relationship between capacitance measurements and anions.A5(prestin) was the positive control and HEK cells transfected by enhanced green fluorescent protein (EGFP) were used as the negative control. The cells number from study was that A2for13, A4for8, A5(prestin) for9, A6for15, A7for11, A9for10, All for8when intracellular and extracellular solution with C1-. All of the HEK-293cells which expressed DNA of A2, A4, A6, A7, A9or All had NLC. In other words, everyone in our study had voltage-dependent property. We compared NLC parameters derived from curve fitting with Boltzmann’s function and found that the largest NLC/Clin was A5(prestin) for7.80±3.27(mean±SD); the smallest one was A2for1.65±0.70; A6was similar as A5(prestin) for6.94±2.30. A6was closed to A5(prestin) in Qmax/Clin, V1/2and z value, also (A5for12.72±4.61fC/pF,-69.79±8.30mV and0.70±0.07, respectively; A6for12.02±3.28fC/pF,-41.74±17.12mV and0.55±0.08, respectively). So A6is the most closely related to A5(prestin) in the voltage-dependent property as suggest in Phylogenetic tree.NLC of A6is closed to A5(prestin) and A6shares similar function with other members of SLC26A family, so we should pay more attention to it because it is a link that may connect A5(prestin) with SLC26A family. Intracellular anions, such as C1-, play a critical role in voltage-dependent motor activity of prestin (A5). When C1-move in and out of prestin, NLC is record. When used sodium pentane sulfonate instead of intracellular Cl", NLC of prestin (A5) will eliminate or reduce. We used the same way to record NLC of HEK-293cells which expressed SLC26A6protein. There was no change when was C1-instead. Parameters (V1/2, z, Qmax/Clin and NLC/Clin) derived from the NLC curve fitting with a Boltzmann function are no statistical significance between these two situation (P=0.718,0.143,0.138and0.534, respectively, Student’s t test). This suggests that Cl" in extracellular solution may be move into protein or NLC is made by other anions. So lmmol DIDS (an anion transporter blocker) was given to cells in order to block channels. NLC of A6was reduced but not disappeared (n=4) after given DIDS. Statistical significance (p<0.05, Student’s t test) was found between DIDS and Cl-or DIDS and Cl--free situation. The data demonstrates that NLC of A6is made by Cl-movements and other anions may play a role in this situation.To further confirm whether other anions except Cl-contribute to the voltage-dependent property, A2was selected to the next plan because NLC of A2is the smallest one in our study. We used a half amount of SO42-to substitute Cl-in the extracellular solution then measured the NLC from13A2-expression HEK-293cells. When added SO42-, NLC/Clin of A2increased and it was twice as much as Cl-groups. There was statistical significance between them (P<0.01, Student’s t test). NLC of All was also record in our study to determine the situation because the human SLC26A11transports SO42-only. We found that NLC/Clin of All (n=11) was significantly increased (P<0.05, Student’s t test) and it was about twice as much as Cl-groups in sulfate solution consistent with A2. We thought that NLC getting to twice in SO42-groups, may be because of the charge with SO42-. In one word, Cl-is not the only voltage sensor of the SLC26A family.Our results suggest that A6is most closely related to A5(prestin) in the SLC26A family; The voltage-dependent property is the common feature of the SLC26A family and NLC of A6is similar as A5(prestin); The voltage-dependent property of the SLC26A family is thought to be generated by voltage-dependent sensors movement in protein driving by voltage; Cl-is not the only voltage sensor of the SLC26A family. In conclusion, SLC26A anion transporters are voltage-gated anion transporters and the sequence which contributes to the voltage sensitive may be the conserved sequence of the SLC26A family. The voltage sensor of the SLC26A anion transporter is not the specific anion.