Reply to Yu, Huang, and Lin: Non-proteolytic HCN2 in the Heart

Thank you for your interest and for your comments on our work. We agree that different laboratories have reported different molecular masses for proteins identified with anti-HCN2 antibodies in lysates and membrane preparations from heart, as well as from brain and from transiently transfected cells. As you point out, for example, several laboratories, including your own, have reported the detection of a protein (or proteins) of ∼100 kDa in extracts from rat and human hearts and transiently transfected cells. All of the papers that we are aware of, however, report the results of experiments using a single antibody (obtained from Alomone Laboratories), and all assume that the identified band is the targeted protein. As with any antibody that has not been rigorously validated (for example against a “knock-out”), it is not possible to conclude that the band (or bands) detected with the anti-HCN2 antibody indeed correspond to the targeted protein. This, however, is typically assumed to be the case and was done in the papers referenced. It is also often the case that antibodies detect multiple bands, some, all, or none of which may correspond to the protein against which the antibody was generated. It is not possible to determine whether multiple protein bands identified with the anti-HCN2 antibodies used in the studies referred to as only portions (cutouts) of the Western blots are displayed in the referenced manuscripts. Clearly, using a knock-out animal (in this case Hcn2−/− animals) as the control for antibody validation studies is the ideal, the “gold standard.” When knock-outs are not available, an alternative (although clearly inferior) approach to validation is to use multiple antibodies targeted against different amino acid sequences in the same protein and also to use different tissue preparations, as we have done here. As discussed in the manuscript, other laboratories have published Western blots in which low molecular mass, ∼60 kDa, proteins were identified with the commercially available (Alomone) anti-HCN2 antibodies. It seems clear that additional experiments will need to be completed to determine the molecular bases of the reported differences in the molecular masses of the proteins identified with anti-HCN2 antibodies in different samples and in different laboratories to resolve the apparent discrepancies in the conclusions derived from the experimental data. A systematic study exploiting multiple different antibodies, preferably validated using Hcn2−/− animals, will likely provide new insights. The error in the labeling of the V½ values in Fig. 5 has been corrected. Also, as noted in the manuscript, the activation curves (in Fig. 5) were derived from measurements of the currents evoked during hyperpolarizing voltage steps and were not determined from tail current measurements. The value given in Fig. 5C for the V½ for If of −97 mV was reported for If in human sinoatrial nodal cells. This is indicated in the figure, and the reference is also provided. It was not our intention to make arguments about the possible role of heteromeric HCN channels in the generation of cardiac If (in any cell type) based on the V½ values determined for heterologously expressed HCN currents. In addition, we certainly recognize that there are large variations in the V½ values reported in the literature for If activation in different cell types and species. The main point in the argument made for a role for heteromeric HCN2/HCN4 channels in the generation of cardiac If concerned the similarity in the kinetics of activation of cardiac If and the currents produced on co-expression of HCN2 and HCN4, compared with the currents produced on expression of either HCN2 or HCN4 alone.