Figure 1.

Electrophysiological properties of cation channels in hereditary spherocytosis red cells. (A) A representative current trace recorded at -Vp = -25mV from an on-cell patch recording from a red cell of patient HS11 with hereditary spherocytosis (HS) carrying the novel heterozygous SPTB missense variant R1255G (Polyphen-2 score 0.999). Identical bath and pipette fluid composition included (in mM) 140 NaCl, 4 KCl, 1 CaCl2, 1 MgCl2, 10 NaHEPES at a final pH of 7.40. On-cell patch currents were recorded by an Axopatch 200b amplifier and digitized by a Digidata 1440A A-D converter (Molecular Devices, Sunnyvale, CA, USA). Seal resistances were 6.0±1.0 GΩ (n=7) in non-HS cells, 5.0±0.8 GΩ (n=14) in HS cells without GsMTx4 in the pipette solution, and 4.8±1.0 GΩ (n=6) in HS cells with GsMTx4 in the pipette solution. Seal duration for recordings on HS cells unexposed to GsMTx4 was 18±11 min. Data were filtered at 500 Hz, digitized at 2 kHz by PClamp and analyzed offline by Clampfit (PCLAMP11, Molecular Devices). (B) Current-voltage relationship of HS11 red cell current measured in a representative on-cell patch, with unitary slope conductance of 21 pS. The current-voltage (I-V) relationship was generated in Clampex (PCLAMP 11, Axon Instruments) with the real-time control window in gap-free mode to record current traces of 10–30 s duration. Test potentials were selected in 25-50 mV increments ranging between a minimum of -100 mV to a maximum of +100 mV. (C) Summary data for NPo calculated from on-cell patch current traces of 5-10 s duration recorded in 16 cells from six HS mutant genotypes and in six cells from three mutant HS genotypes in the additional presence of GsMTx4 (1 μM) in the pipette fluid. NPo values recorded in seven non-HS red cells from four normal individuals (AA) are also shown. *P<0.05 for the t-test comparing normal to HS cells, and for the Mann-Whitney test comparing HS cells in the presence versus absence of GsMTx4.