Philemon S. Yang, Badr A. Alseikhan, Hakim Hiel, Masayuki X. Mori, Wanjun Yang, Paul A. Fuchs, and David T. Yue

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1 Supplemental Data Switching of Ca 2+ -dependent inactivation of Ca V 1.3 channels by calcium binding proteins of auditory hair cells Yang et al (2006) J Neurosci 26: Philemon S. Yang, Badr A. Alseikhan, Hakim Hiel, Masayuki X. Mori, Wanjun Yang, Paul A. Fuchs, and David T. Yue 1. Cell-to-cell fluctuations in current reflect variations in channel number For the analysis involving plots of CDI strength versus current amplitude, an underlying assumption is that cell-to-cell fluctuations in current amplitude reflect variations in channel number, rather than changes in channel open probability. One straightforward means of testing this assumption is to determine both the maximal gating charge (Q max ), and the maximal ioniccurrent slope conductance (G max ) within individual cells. Consider that Q max = N channel q max, where N channel is the number of channels in a given cell, and q max is the maximal gating charge per channel. Also consider that G max = N channel g max P max, where g max is the single-channel conductance at saturating voltages, and P max is the open probability at saturating depolarization. It follows that if G max is plotted as a function of Q max (on a cell-by-cell basis), the resulting relationship will form a straight line, only if the above underlying assumption holds true. Moreover, if such a straight line is encountered, then the slope of that line should be proportional to P max, because g max and q max are unlikely to show appreciable variation among channels. In prior work, we have extensively employed this approach for dissecting G-protein modulation of N-type channels (Agler et al., 2005). Application of this same approach to Ca V 1.3 channels is shown in Figure S1. Q max is gauged by integrating the transient current seen upon depolarization to the reversal potential (A, green-shaded area), while G max is determined by linear regression of the saturating phase of I-V relations (B, red segment) derived from ramp depolarization of the same cells. Plotting these two entities approximates a straight line (C), with slope ~1.78 ns/fc. These results thereby support the underlying assumption that cell-to-cell fluctuations in current amplitude reflect variations in channel number. Figure S1 G-Q analysis of Ca V 1.3 channels. Currents recorded in standard solutions with 10 mm Ba 2+ as charge carrier. A, Currents obtained at reversal potential filtered at 5 khz, and sampled at 100 khz. B, I-V relations determined from ramp voltage depolarization protocols. C, G max versus Q max plots, with each symbol corresponding to a single cell. Supplemental Data for Yang et al, page 1 of 6

2 2. Detection of CaBP transcripts in rat organ of Corti by RT-PCR analysis Figure S2 CaBP molecules and annealing sites of PCR primers. Top shows schematic of generic CaBP, with EF hands represented as gray rectangles. Amino terminus at left. Middle shows schematic specific for CaBP4, with approximate locations of PCR primers used for RT-PCR. Bottom shows analogous information for CaBP1. Details of RT-PCR analysis were described in the Experimental Procedures of the main text. The PCR products for CaBP experiments were ligated into bacterial plasmids, which were cloned into individual bacterial colonies. PCR inserts from individual colonies were sequenced, and the results are displayed on the following two pages. Supplemental Data for Yang et al, page 2 of 6

3 Figure S3 indicates that, at the amino acid level, PCR inserts for the CaBP4 RT-PCR experiments (capital letters) predicted 100% identity to previously published rat CaBP4 sequence (lower-case letters: GenBank, XM ). At the nucleotide level, there were three silent differences (highlighted in yellow). The red boxes explicitly delineate primer annealing segments. Figure S3 Sequence alignment of RT-PCR product (capital letters) and CaBP4 (lower-case letters). PCR primer locations explicitly boxed in red. EF-hand binding loops underlined. Supplemental Data for Yang et al, page 3 of 6

4 Figure S4 indicates that, at the amino acid level, PCR inserts for the CaBP1 RT-PCR experiments (capital letters) predicted 100% identity to previously published rat CaBP1 sequence (lower-casel letters: GenBank, NM ). At the nucleotide level, there were no differences, except for silent mutations in the primers themselves (highlighted in yellow). The red boxes explicitly delineate primer annealing segments. Figure S4 Sequence alignment of RT-PCR product (capital letters) and rat CaBP1 (lower-case letters). PCR primer locations explicitly boxed in red. EF-hand binding loops underlined. Supplemental Data for Yang et al, page 4 of 6

5 3. Preadsorption controls for CaBP antibody stains of slices from rat organ of Corti Figure S5 shows the preadsorption control for the CaBP immunostains shown in Figures 5B and 5C. Methods were identical to those used for Figure 5 (see Experimental Procedures), except that either CaBP1 or CaBP4 antibody (at the working dilution of 1:3000) was mixed in the blocking buffer with their generating peptides at concentrations of 1.5 and 2.9 ìm, respectively. As well, NF200 antibody (at the working dilution of 1:1000) was added to the mixture, so as to visualize the afferent nerve fibers contacting sensory hair cells. These peptide/antibody mixtures were incubated at room temperature for 2 hours prior to their application to cochlear tissue sections. Of note, the control experiments were run in parallel to their respective positive immunostaining experiments (in Figure 5). The persistence of the red signal from neurofilament, concurrent with the elimination of green signal from CaBP antibody, demonstrates the specificity of green CaBP signal in Figure 5. For immunocytochemistry and western blot analysis of retinal tissue, these same antibodies have previously been shown to label specifically CaBP1 (Haeseleer et al., 2000) and CaBP4 molecules (Haeseleer et al., 2004). Figure S5 Preadsorption controls for CaBP immunostains of rat organ of Corti. The top panel shows the control corresponding to CaBP1 staining in Figure 5B, bottom, for a P28 rat. The bottom panel shows the control corresponding to the CaBP4 staining in Figure 5C, bottom, for a P28 rat. Format as in Figures 5B and 5C. Note maintained red signal from NF200, despite elimination of green signal for CaBP. 4. CaBP1 staining is disjoint from ChAT immunoreactivity at efferent nerve terminals Given the proximity of Deiters cell processes and efferent nerve terminals, it was conceivable that the CaBP1 immunoreactivity in Figures 5B and 5D (P28) of the main text actually originated from these nerve terminals, rather than from Deiters cells. Figure S6 investigates this issue, by co-staining a rat P28 section for both CaBP1 (green) and ChAT (red, choline acetyltransferase), the latter of which is a specific marker for efferent nerve terminals. The disjoint nature of CaBP1 and ChAT signals confirms that CaBP1 legitimately resides in Deiters cell processes. In particular, CaBP1-positive structures enveloped the ChAT-positive endings, echoing the Deiters cell chalice that envelops the synaptic poles of OHCs (Furness et al., 2002). Methods were as used for Figure 5 (of the main text, see Experimental Procedures), except that ChAT antibody (at Supplemental Data for Yang et al, page 5 of 6

6 the working dilution of 1:1000) was added to the staining mixture (instead of anti NF200), so as to visualize the efferent nerve fiber contact with outer hair cells. Figure S6 Co-staining of P28 rat organ of Corti section by anti-cabp1 and anti-chat antibodies. The image shows a row of three outer hair cells. Overlay of brightfield, anti-cabp1 immunofluorescence (green), and anti- ChAT immunofluorescence (red) signals. Green fluorescence indicates characteristic CaBP1 staining of Deiters-cell chalices (open arrows), as well as OHC cuticular plates (open circles). Anti-ChAT immunofluorescence (red) indicates efferent nerve terminals, which is clearly different from the CaBP1 (green) present in Deiters cells. CaBP1-P28 5. References Agler HL, Evans J, Tay LH, Anderson MJ, Colecraft HM, Yue DT (2005) G protein-gated inhibitory module of N-type (Ca V 2.2) Ca 2+ channels. Neuron 46: Furness DN, Hulme JA, Lawton DM, Hackney CM (2002) Distribution of the glutamate/aspartate transporter GLAST in relation to the afferent synapses of outer hair cells in the guinea pig cochlea. J Assoc Res Otolaryngol 3: Haeseleer F, Imanishi Y, Maeda T, Possin DE, Maeda A, Lee A, Rieke F, Palczewski K (2004) Essential role of Ca 2+ -binding protein 4, a Ca V 1.4 channel regulator, in photoreceptor synaptic function. Nat Neurosci 7: Haeseleer F, Sokal I, Verlinde CL, Erdjument-Bromage H, Tempst P, Pronin AN, Benovic JL, Fariss RN, Palczewski K (2000) Five members of a novel Ca 2+ -binding protein (CaBP) subfamily with similarity to calmodulin. J Biol Chem 275: Supplemental Data for Yang et al, page 6 of 6